In vitro model for pathological or physiologic conditions

ABSTRACT

The present invention generally relates to in vitro methods for mimicking in vivo pathological or physiologic conditions. The methods comprise applying shear forces to a cell type or cell type plated on a surface within a cell culture container. Methods for testing drugs or compounds in such systems are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/599,821, filed on May 19, 2017, which is a continuation of U.S.patent application Ser. No. 13/866,017, filed on Apr. 18, 2013 and nowissued as U.S. Pat. No. 9,658,211, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/724,864, filed on Nov. 9,2012 and U.S. Provisional Patent Application Ser. No. 61/635,118, filedon Apr. 18, 2012. Each of the above-cited applications is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to in vitro methods formimicking in vivo pathological or physiologic conditions. The presentinvention also relates to methods for testing drugs or compounds in suchsystems.

BACKGROUND OF THE INVENTION

Conventional in vitro models of pathological or physiological conditionsgenerally involve culturing one or more cell types under staticconditions. However, such models typically require the addition of oneor more factors in concentrations much higher than those observed invivo in the pathological or physiological condition. For example, inorder to maintain hepatocytes in static tissue culture, insulin andglucose must be added to the culture media in concentrationssignificantly higher than the concentrations observed in vivo in healthyindividuals (by approximately 2 to 4-fold for glucose, and about10,000-fold to 40,000-fold for insulin). Similarly, in conventionalstatic monocultures of endothelial cells used to model thrombosis,significantly elevated levels of TNFα as compared to those observed inhuman circulating blood are required to induce fibrin deposition.

Furthermore, the conventional systems often do not exhibit responses todrugs or compounds at concentrations that induce the response in vivo,instead requiring much higher concentrations of the drug or compound toinduce the same response.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a method of mimickinga pathological condition in vitro. The method comprises adding a culturemedia to a cell culture container, adding at least one factor to theculture media, plating at least one cell type on at least one surfacewithin the cell culture container, and applying a shear force upon theat least one plated cell type. The shear force results from flow of theculture media induced by a flow device. The flow mimics flow to whichthe at least one cell type is exposed in vivo in the pathologicalcondition. The concentration of the factor in the culture media can bewithin the in vivo concentration range of the factor observed in thepathological condition. Alternatively, the concentration of the factorin the culture media can be within the concentration range of the factorthat would result in vivo from administration of a drug or a compound.

Another aspect of the present invention is an in vitro method of testinga drug or a compound for an effect on a pathological condition. Themethod comprises mimicking the pathological condition, adding a drug ora compound to the culture media, and applying the shear force upon theat least one plated cell type exposed to the drug or the compound. Achange in the at least one plated cell type, in the presence of the drugor the compound, indicates that the drug or the compound has an effecton the pathological condition. In this in vitro method of testing a drugor compound, the pathological condition can be mimicked by the in vitromethod of mimicking a pathological condition as described above.

The present invention also provides an in vitro method of testing a drugor compound for an effect. The method comprises adding a culture mediato a cell culture container, plating at least one cell type on at leastone surface within the cell culture container, adding a drug or acompound to the culture media, and applying a shear force upon the atleast one plated cell type exposed to the drug or the compound. Theconcentration of the drug or the compound in the culture media is withinthe concentration range of the drug or the compound that achieves theeffect in vivo. The shear force results from flow of the culture mediainduced by a flow device. The flow mimics flow to which the at least onecell type is exposed in vivo. A change in the at least one plated celltype, in the presence of the drug or the compound, indicates that thedrug or the compound has the effect. The effect can be, for example, aneffect on a physiologic condition or an effect on a pathologicalcondition.

Another aspect of the present invention is a method of mimicking aphysiologic condition in vitro. The method comprises adding a culturemedia to a cell culture container, adding at least one factor to theculture media, plating at least one cell type on at least one surfacewithin the cell culture container, and applying a shear force upon theat least one plated cell type. The shear force results from flow of theculture media induced by a flow device. The flow mimics flow to whichthe at least one cell type is exposed in vivo in the physiologiccondition. The concentration of the factor in the culture media can bewithin the in vivo concentration range of the factor observed in thephysiologic condition. Alternatively, the concentration of the factor inthe culture media can be within the concentration range of the factorthat would result in vivo from administration of a drug or a compound.

The present invention is also directed to an in vitro method of testinga drug or a compound for an effect on a physiologic condition. Themethod comprises mimicking the physiologic condition, adding a drug or acompound to the culture media, and applying the shear force upon the atleast one plated cell type exposed to the drug or the compound. A changein the at least one plated cell type, in the presence of the drug or thecompound, indicates that the drug or the compound has an effect on thephysiologic condition. In this in vitro method of testing a drug orcompound, the physiologic condition can be mimicked by the in vitromethod of mimicking a physiologic condition as described above.

Another aspect of the invention is a method of mimicking a pathologicalor physiologic condition of the liver in vitro. The method comprisesadding a culture media to a cell culture container, adding at least onefactor to the culture media, plating at least one hepatic cell type onat least one surface within the cell culture container, and applying ashear force upon the at least one plated hepatic cell type. The shearforce results from flow of the culture media induced by a flow device.The flow mimics flow to which the at least one hepatic cell type isexposed in vivo in the pathological or physiologic condition. Theconcentration of the factor in the culture media for mimicking thepathological condition can be within the in vivo concentration range ofthe factor observed in the pathological condition. Alternatively, theconcentration of the factor in the culture media can be within theconcentration range of the factor that would result in vivo fromadministration of a drug or a compound. As a further alternative, theconcentration of the factor in the culture media can be capable ofmaintaining the mimicked pathological condition in vitro for a period oftime under the shear force, the same concentration of factor beingincapable of maintaining the mimicked pathological condition in vitrofor the period of time in the absence of the shear force. Theconcentration of the factor in the culture media for mimicking thephysiologic condition can be within the in vivo concentration range ofthe factor observed in the physiologic condition. Alternatively, theconcentration of the factor in the culture media can be within theconcentration range of the factor that would result in vivo fromadministration of a drug or a compound. As a further alternative, theconcentration of the factor in the culture media can be capable ofmaintaining the mimicked physiologic condition in vitro for a period oftime under the shear force, the same concentration of factor beingincapable of maintaining the mimicked physiologic condition in vitro forthe period of time in the absence of the shear force.

The present invention also provides an in vitro method of testing a drugor a compound for an effect on a pathological or physiologicalcondition. The method comprises mimicking the pathological orphysiological condition, adding a drug or a compound to the culturemedia, and applying the shear force upon at least one plated hepaticcell type exposed to the drug or the compound. A change in the at leastone plated hepatic cell type, in the presence of the drug or thecompound, indicates that the drug or the compound has an effect on thepathological or physiological condition. In this in vitro method oftesting a drug or compound, the pathological condition can be mimickedby the in vitro method of mimicking a pathological or physiologicalcondition as described in the immediately preceding paragraph.

Another aspect of the invention is directed to a method of mimicking apathological or physiologic condition of the liver in vitro. The methodcomprises adding a culture media to a cell culture container, depositingat least one extracellular matrix component on a surface within the cellculture container, plating hepatocytes on the at least one extracellularmatrix component, and indirectly applying a shear force upon the atleast one extracellular matrix component and the hepatocytes. The shearforce results from flow of the culture media induced by a flow device.The flow mimics flow to which the hepatocytes are exposed in vivo in thepathological or physiologic condition.

The invention also provides another method of mimicking a pathologicalor physiologic condition of the liver in vitro. The method comprisesadding a culture media to a cell culture container and platinghepatocytes on a first surface of a porous membrane. The porous membraneis suspended in the cell culture container such that the first surfaceis proximal and in spaced relation to a bottom surface of the container,thereby defining within the container a lower volume comprising thehepatocytes and an upper volume comprising a second surface of theporous membrane. A shear force is applied upon the second surface of theporous membrane in the upper volume of the container, the shear forceresulting from flow of the culture media induced by a flow device. Theflow mimics flow to which the hepatocytes are exposed in vivo in thepathological or physiologic condition. The flow device comprises a bodyadapted for being positioned in the culture media in the upper volume ofthe container and a motor adapted to rotate the body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts an exemplary protocol for a thrombosis assay performedunder static culture conditions.

FIGS. 1B and 1C show exemplary fluorescent microscopy results from athrombosis assay performed under static culture conditions.

FIG. 2A illustrates an exemplary protocol for application of atheroproneor atheroprotective hemodynamic flow to co-cultures of endothelial cellsand smooth muscle cells.

FIG. 2B shows exemplary heat maps of gene expression of genes relevantto thrombosis in endothelial cells and smooth muscle cells grown underatheroprone or atheroprotective hemodynamic flow conditions.

FIG. 3A shows an exemplary protocol for a thrombosis assay performedunder hemodynamic culture conditions.

FIG. 3B shows exemplary fluorescent microscopy results from a thrombosisassay performed under hemodynamic culture conditions.

FIG. 4A depicts an exemplary protocol for a thrombosis assay performedunder hemodynamic culture conditions, with continued application ofshear stress during clot formation.

FIG. 4B depicts exemplary fluorescent microscopy results from athrombosis assay performed under hemodynamic culture conditions, withcontinued application of shear stress during clot formation.

FIG. 5 shows a protocol for assays wherein co-cultures of endothelialcells and smooth muscle cells are subjected to hemodynamicpreconditioning, followed by treatment with one or more factors.

FIGS. 6A-F depict exemplary gene expression data for assays using oxLDL.

FIGS. 7A-E illustrate changes in gene expression in response to oxLDL.

FIGS. 8A and 8B depict exemplary data showing NFκB activity in responseto oxLDL.

FIG. 9 shows exemplary differential gene regulation data for cellstreated with oxLDL.

FIG. 10 shows exemplary data showing gene expression in response toTNFα.

FIGS. 11A-B show exemplary gene expression data in response to treatmentwith oxLDL and TNFα.

FIGS. 12A-C depict exemplary data showing NFκB activity and changesexpression of genes involved in inflammatory signaling in response totreatment with glucose and TNFα.

FIGS. 13A-B show exemplary gene array data for endothelial cells andsmooth muscle cells treated with angiotensin II under hemodynamicconditions.

FIGS. 14A-B show exemplary gene array data for endothelial cells andsmooth muscle cells treated with aldosterone under hemodynamicconditions.

FIG. 15A is a schematic drawing of a liver sinusoid.

FIG. 15B depicts the cone-and-plate device and the application ofindirect shear forces to hepatocytes.

FIG. 15C depicts the plating configuration of hepatocytes in the invitro liver model.

FIGS. 16A-F are exemplary fluorescent microscopy images of hepatocytescultured under static conditions or in the presence of controlledhemodynamics.

FIG. 17A is an exemplary fluorescent microscopy image of hepatocytescultured under controlled hemodynamics.

FIG. 17B is an exemplary fluorescent microscopy image of in vivo liver.

FIG. 17C shows exemplary transmission electron microscopy images ofhepatocytes cultured under controlled hemodynamics.

FIGS. 18A-B show exemplary data for urea and albumin secretion inhepatocytes cultured under static conditions or controlled hemodynamics.

FIGS. 19A-D show exemplary metabolic gene expression data forhepatocytes cultured under static conditions or controlled hemodynamics.

FIGS. 20A-B show exemplary cytochrome p450 activity data for hepatocytescultured under static conditions or controlled hemodynamics.

FIG. 20C is an exemplary fluorescent microscopy image from an assay fortransporter activity in hepatocytes cultured under controlledhemodynamics.

FIG. 21 shows exemplary gene expression data for the in vitro fattyliver model.

FIG. 22 shows exemplary gene expression data for the in vitro fattyliver model.

FIG. 23 is a perspective of the clip that mounts on the cell culturedish and secures inflow and outflow tubing to perfuse the upper andlower volumes.

FIGS. 24A and 24B show exemplary plating configurations of endothelialcells and smooth muscle cells within the cell culture container.

FIGS. 25A-B show exemplary fluorescent microscopy images of hepatocytescultured under healthy conditions or conditions that mimic fatty liverdisease.

FIG. 26 shows a transmission electron microscopy image of rathepatocytes cultured under high glucose/high insulin conditions.

FIGS. 27A-B show exemplary results from assays measuring total lipidsand total triglycerides in hepatocytes cultured under healthy conditionsor conditions that mimic fatty liver disease.

FIG. 28AB show exemplary gene expression data for hepatocytes culturedunder healthy conditions or conditions that mimic fatty liver disease.

FIGS. 29A-B provide exemplary metabolic gene expression data andcytochrome p450 activity data for hepatocytes cultured under healthyconditions or conditions that mimic fatty liver disease.

FIGS. 30A-3C show exemplary fluorescent microscopy images fromhepatocytes cultured under healthy conditions or under conditions thatmimic fatty liver disease, in the presence or absence of pioglitazone.

FIG. 31 provides exemplary results from an assay measuring totaltriglycerides in hepatocytes cultured under healthy conditions or underconditions that mimic fatty liver disease, in the presence or absence ofpioglitazone.

FIG. 32 provides exemplary metabolic gene expression data forhepatocytes cultured under healthy conditions or under conditions thatmimic fatty liver disease, in the presence or absence of pioglitazone.

Corresponding reference characters indicate corresponding partsthroughout the drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides in vitro methods for mimicking an in vivopathological or physiologic condition. Unlike static models currentlyused as the standard in vitro models by the pharmaceutical andbiopharmaceutical industries, the methods of the invention apply shearforces to cultured cells and replicate an in vivo pathological orphysiological condition using in vivo pathological or physiologicconcentrations of various factors. For example, an in vitro liver modelhas been discovered in which hepatocytes can be maintained at in vivophysiologic concentrations of insulin and glucose that are significantlydecreased as compared to the concentrations used in the standard staticmodel. It has further been discovered that when higher concentrations ofinsulin and glucose are used in such a model, the hepatocytes exhibitnumerous hallmarks of fatty liver disease.

The present invention is also directed to a method of mimicking apathological condition in vitro. The method comprises adding a culturemedia to a cell culture container, adding at least one factor to theculture media, plating at least one cell type on at least one surfacewithin the cell culture container, and applying a shear force upon theat least one plated cell type. The shear force results from flow of theculture media induced by a flow device. The flow mimics flow to whichthe at least one cell type is exposed in vivo in the pathologicalcondition.

The concentration of the factor in the culture media can be within thein vivo concentration range of the factor observed in the pathologicalcondition. Alternatively, the concentration of the factor in the culturemedia can be within the concentration range of the factor that wouldresult in vivo from administration of a drug or a compound.

To confirm that the in vivo pathological condition is mimicked, a changein a level of a marker of the pathological condition can be comparedbetween the method of the invention and the same method in the absenceof application of the shear force. The level of the marker in the atleast one plated cell type or in the culture media upon application ofthe shear force is compared to the level of the marker in the at leastone plated cell type or in the culture media in the absence ofapplication of the shear force. For example, if a marker is known to beassociated with a pathological condition and its concentration is knownto increase in the serum when the condition is present in vivo, anincrease in the level of the marker in the culture media of the methodof the invention with application of the shear force as compared to thelevel of the marker in the culture media in the absence of applicationof the shear force confirms that the in vivo pathological condition ismimicked by the in vitro method of the invention.

Pathological conditions, effects on the pathological conditions,physiologic conditions, flow devices, hemodynamic patterns, cell types,and cell culture media including factors added to the cell culture mediafor use in the methods of the invention are described in detail below,following the description of the various methods of the invention.

The present invention is also directed to an in vitro method of testinga drug or a compound for an effect on a pathological condition. Themethod comprises mimicking the pathological condition, adding a drug ora compound to the culture media, and applying the shear force upon theat least one plated cell type exposed to the drug or the compound. Achange in the at least one plated cell type, in the presence of the drugor the compound, indicates that the drug or the compound has an effecton the pathological condition.

In this in vitro method of testing a drug or compound, the pathologicalcondition can be mimicked by the in vitro method of mimicking apathological condition as described above.

The pathological condition of the in vitro method of testing a drug orcompound can also be mimicked by plating primary cells or immortalizedcells from a subject or subjects having the pathological condition, andculturing the cells in cell culture media.

The present invention is also directed to a method of mimicking aphysiologic condition in vitro. The method comprises adding a culturemedia to a cell culture container, adding at least one factor to theculture media, plating at least one cell type on at least one surfacewithin the cell culture container, and applying a shear force upon theat least one plated cell type. The shear force results from flow of theculture media induced by a flow device. The flow mimics flow to whichthe at least one cell type is exposed in vivo in the physiologiccondition.

The concentration of the factor in the culture media can be within thein vivo concentration range of the factor observed in the physiologiccondition. Alternatively, the concentration of the factor in the culturemedia can be within the concentration range of the factor that wouldresult in vivo from administration of a drug or a compound.

To confirm that the in vivo physiologic condition is mimicked, a changein a level of a marker of the physiologic condition can be comparedbetween the method of the invention and the same method in the absenceof application of the shear force. The level of the marker in the atleast one plated cell type or in the culture media upon application ofthe shear force is compared to the level of the marker in the at leastone plated cell type or in the culture media in the absence ofapplication of the shear force. For example, if a marker is known to beassociated with a physiologic condition and its concentration is knownto increase in the serum when the condition is present in vivo, anincrease in the level of the marker in the culture media of the methodof the invention with application of the shear force as compared to thelevel of the marker in the culture media in the absence of applicationof the shear force confirms that the in vivo physiologic condition ismimicked by the in vitro method of the invention.

The present invention is also directed to an in vitro method of testinga drug or a compound for an effect on a physiologic condition. Themethod comprises mimicking the physiologic condition, adding a drug or acompound to the culture media, and applying the shear force upon the atleast one plated cell type exposed to the drug or the compound. A changein the at least one plated cell type, in the presence of the drug or thecompound, indicates that the drug or the compound has an effect on thephysiologic condition.

In this in vitro method of testing a drug or compound, the physiologiccondition can be mimicked by the in vitro method of mimicking aphysiologic condition as described above.

The physiologic condition of this in vitro method of testing a drug orcompound can also be mimicked by plating primary cells or immortalizedcells, and culturing the cells in cell culture media. The primary orimmortalized cells are described in detail below.

The present invention also relates to an in vitro method of testing adrug or a compound for an effect. The method comprises adding a culturemedia to a cell culture container, plating at least one cell type on atleast one surface within the cell culture container, adding a drug or acompound to the culture media, and applying a shear force upon the atleast one plated cell type exposed to the drug or the compound. Theconcentration of the drug or the compound in the culture media is withinthe concentration range of the drug or the compound that achieves theeffect in vivo. The shear force results from flow of the culture mediainduced by a flow device. The flow mimics flow to which the at least onecell type is exposed in vivo. A change in the at least one plated celltype, in the presence of the drug or the compound, indicates that thedrug or the compound has the effect.

The effect can be an effect on a pathological condition. Alternatively,the effect can be an effect on a physiologic condition. Further effectsare described in detail below.

In any of the methods of the invention, the method can further compriseanalyzing the cell culture media for cytokine secretion, chemokinesecretion, humoral factor secretion, microparticle secretion, growthfactor secretion, shedding of a protein from the cellular surface, ametabolite of a compound, an immune cell, nitric oxide secretion, avasodilator protein, a vasoconstrictive protein, miRNA, a secretedprotein, or a secreted biological substance. The cell culture media canbe analyzed for nitric oxide secretion by measuring nitrate or nitriteconcentration.

When the cell culture media is analyzed for shedding of a protein fromthe cellular surface, the protein can comprise a vascular cell adhesionmolecule (VCAM), E-selectin, or an intracellular adhesion molecule(ICAM).

In any of the methods of the invention, the method can further comprisethe step of culturing the cell type or cell types.

In any of the methods of the invention wherein a drug or compound hasbeen added to the culture media, the method can further comprise thestep of comparing at least one of the cell types after applying theshear force for a period of time wherein the media includes the drug orthe compound to the at least one of the cell types after applying theshear force for the period of time wherein the media does not includethe drug or the compound, to determine the effect of the drug orcompound on the at least one of the cell types.

In Vitro Liver Models

When a drug or a compound is tested for an effect on a healthy liver,the factors comprise insulin and glucose, hepatocytes are plated on thesurface within the cell culture container, and the shear force isapplied indirectly to the plated hepatocytes.

For example, the hepatocytes can be plated on a first surface of aporous membrane. The porous membrane is then suspended in the cellculture container such that the first surface is proximal and in spacedrelation to a bottom surface of the cell culture container, therebydefining within the cell culture container a lower volume and an uppervolume. The lower volume comprises the hepatocytes and the upper volumecomprises a second surface of the porous membrane. The shear force isapplied to the second surface of the porous membrane in the upper volumeof the container.

In any of the methods of the invention, use of a porous membranesuspended in the cell culture container is preferred in plating thecells. When shear force is applied to plated cells or to the surface ofthe porous membrane (e.g., when the shear is applied on a surface of themembrane absent plated cells), the shear force can enable the cellculture media to perfuse from the upper volume to the lower volume. Suchperfusion favorably impacts transport of factors from the upper volumeto the lower volume, or vice versa.

The invention is also directed to a method of mimicking a pathologicalor physiologic condition of the liver in vitro. The method comprisesadding a culture media to a cell culture container, adding at least onefactor to the culture media, plating at least one hepatic cell type onat least one surface within the cell culture container, and applying ashear force upon the at least one plated hepatic cell type. The shearforce results from flow of the culture media induced by a flow device.The flow mimics flow to which the at least one hepatic cell type isexposed in vivo in the pathological or physiologic condition.

In this method, the concentration of the factor in the culture media formimicking the pathological condition can be within the in vivoconcentration range of the factor observed in the pathologicalcondition. Alternatively, in this method, the concentration of thefactor in the culture media for mimicking the pathological condition canbe within the concentration range of the factor that would result invivo from administration of a drug or a compound. As a furtheralternative, in this method, the concentration of the factor in theculture media for mimicking the pathological condition can be capable ofmaintaining the mimicked pathological condition in vitro for a period oftime under the shear force, the same concentration of factor beingincapable of maintaining the mimicked pathological condition in vitrofor the period of time in the absence of the shear force.

In this method, the concentration of the factor in the culture media formimicking the physiologic condition can be within the in vivoconcentration range of the factor observed in the physiologic condition.Alternatively, in this method, the concentration of the factor in theculture media for mimicking the physiologic condition can be within theconcentration range of the factor that would result in vivo fromadministration of a drug or a compound. As a further alternative, inthis method, the concentration of the factor in the culture media formimicking the physiologic condition can be capable of maintaining themimicked physiologic condition in vitro for a period of time under theshear force, the same concentration of factor being incapable ofmaintaining the mimicked physiologic condition in vitro for the periodof time in the absence of the shear force.

In this method, a change in a level of a marker of the pathological orphysiologic condition in the at least one plated hepatic cell type or inthe culture media upon application of the shear force, as compared tothe level of the marker in the at least one plated hepatic cell type orin the culture media in the absence of application of the shear forceconfirms mimicking of the pathological or physiologic condition.

The present invention is also directed to an in vitro method of testinga drug or a compound for an effect on a pathological or physiologicalcondition. The method comprises mimicking the pathological orphysiological condition, adding a drug or a compound to the culturemedia, and applying the shear force upon at least one plated hepaticcell type exposed to the drug or the compound. A change in the at leastone plated hepatic cell type, in the presence of the drug or thecompound, indicates that the drug or the compound has an effect on thepathological or physiological condition.

In this in vitro method of testing a drug or compound, the pathologicalcondition can be mimicked by the in vitro method of mimicking apathological or physiological condition as described directly above.

The pathological or physiological condition of the in vitro method oftesting a drug or compound can also be mimicked by plating primary cellsor immortalized cells from a subject or subjects having the pathologicalcondition, and culturing the cells in cell culture media.

The invention is also directed to a method of mimicking a pathologicalor physiologic condition of the liver in vitro. The method comprisesadding a culture media to a cell culture container, depositing at leastone extracellular matrix component on a surface within the cell culturecontainer, plating hepatocytes on the at least one extracellular matrixcomponent, and indirectly applying a shear force upon the at least oneextracellular matrix component and the hepatocytes. The shear forceresults from flow of the culture media induced by a flow device. Theflow mimics flow to which the hepatocytes are exposed in vivo in thepathological or physiologic condition.

In methods of the invention in which hepatic cells are plated on aporous membrane, at least one extracellular matrix component can beplated on a first surface of the porous membrane and the hepatic cellscan subsequently be plated on the at least one extracellular matrixcomponent. Optionally, nonparenchymal hepatic cells (e.g., sinusoidalendothelial cells) can be plated on the second surface of the porousmembrane, and the shear stress applied to the nonparenchymal hepaticcells.

In the methods of the invention involving the deposition of anextracellular matrix component, for example, the at least oneextracellular matrix component can be deposited on a first surface of aporous membrane. The hepatic cell type (e.g., hepatocytes) issubsequently plated on the at least one extracellular matrix component.The porous membrane is suspended in the cell culture container such thatthe first surface is proximal and in spaced relation to a bottom surfaceof the cell culture container, thereby defining within the cell culturecontainer a lower volume and an upper volume. The lower volume comprisesat least one extracellular matrix component and the hepatic cell type(e.g., hepatocytes), and the upper volume comprises a second surface ofthe porous membrane. The shear force is applied to the second surface ofthe porous membrane in the upper volume of the container. Optionally,nonparenchymal hepatic cells (e.g., sinusoidal endothelial cells) can beplated on the second surface of the porous membrane, and the shearstress applied to the nonparenchymal hepatic cells.

The invention also provides another method of mimicking a pathologicalor physiologic condition of the liver in vitro. The method comprisesadding a culture media to a cell culture container, and platinghepatocytes on a first surface of a porous membrane. The porous membraneis suspended in the cell culture container such that the first surfaceis proximal and in spaced relation to a bottom surface of the container,thereby defining within the container a lower volume comprising thehepatocytes and an upper volume comprising a second surface of theporous membrane. A shear force is applied upon the second surface of theporous membrane in the upper volume of the container, the shear forceresulting from flow of the culture media induced by a flow device. Theflow mimics flow to which the hepatocytes are exposed in vivo in thepathological or physiologic condition. The flow device comprises a bodyadapted for being positioned in the culture media in the upper volume ofthe container and a motor adapted to rotate the body. Preferably, thebody has a conical surface. It is also preferred that the flow device isadapted for positioning the conical surface of the body in the containerand in contact with the cell culture media.

This method can further comprise plating nonparenchymal hepatic cells onthe second surface of the porous membrane, wherein the shear stress isapplied to the nonparenchymal hepatic cells. The nonparenchymal hepaticcells can comprise sinusoidal endothelial cells, hepatic stellate cells,Kupffer cells, or combinations thereof.

In the in vitro methods for mimicking a pathological or physiologiccondition of the liver, a change in a level of a marker of thepathological or physiologic condition can be compared in the inventivemethod to the same method in the absence of application of the shearforce. A change in the level of the marker in any of the hepatic cellsor in the culture media upon application of the shear force as comparedto the level of the marker in the hepatic cells or in the culture mediain the absence of application of the shear force confirms mimicking ofthe pathological or physiologic condition. For example, a change in thelevel of the marker in the hepatocytes or nonparenchymal hepatic cellsor in the culture media upon application of the shear force as comparedto the level of the marker in the hepatocytes or nonparenchymal hepaticcells or in the culture media in the absence of application of the shearforce confirms mimicking of the pathological or physiologic condition.

Pathological Conditions and Associated Factors

The pathological conditions include, but are not limited to, advancedinflammation, atherosclerosis, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, hypertension, hypertensiveencephalopathy, hypertensive retinopathy, fatty liver disease,hypertension, heart failure, stroke, Marfan syndrome, carotidintima-medial thickening, atrial fibrillation, kidney disease, pulmonaryfibrosis, chronic obstructive pulmonary disease, hyperlipidemia,hypercholesterolemia, diabetes, atherosclerotic plaque rupture,atherosclerotic plaque erosion, thoracic aortic aneurysm, cerebralaneurysm, abdominal aortic aneurysm, cerebral aneurysm, pulmonary arterydisease, pulmonary hypertension, peripheral artery disease, deep veinthrombosis, vascular restenosis, vascular calcification, myocardialinfarction, obesity, hypertriglyceridemia, hypoalphalipoproteinemia,fatty liver disease, hepatitis C, hepatitis B, liver fibrosis, bacterialinfection, viral infection, cirrhosis, liver fibrosis, andalcohol-induced liver disease.

The pathological condition can comprise an anatomical condition, such asatrophy, calculi, choristoma, pathologic constriction, pathologicdilation, diverticulum, hypertrophy, polyps, prolapse, rupture, anarteriovenous fistula, or an appendage (e.g., left atrial appendage).

For a vascular pathological condition, endothelial cells, smooth musclecells, or endocardial cells can be plated on the surface within the cellculture container, and the shear force applied upon the platedendothelial cells, smooth muscle cells, or endocardial cells.

For a vascular pathological condition, the factor can comprise oxidizedlow-density lipoprotein (oxLDL), tumor necrosis factor-α (TNFα),glucose, tissue growth factor-β (TGF-β), an elastin degradation product,elastase, vitamin D, an inorganic phosphate, leptin, adiponectin,apelin, aldosterone, angiotensin II, a triglyceride, high-densitylipoprotein (HDL), oxidized high-density lipoprotein (oxHDL), atriglyceride-rich lipoprotein, low-density lipoprotein (LDL), insulin, afatty acid, or a combination thereof.

The triglyceride-rich lipoprotein can comprise very low-densitylipoprotein (vLDL), a vLDL remnant, a chylomicron, or a chylomicronremnant.

For a vascular pathological condition where a porous membrane is used,endocardial cells can be plated on a first surface of a porous membrane.The porous membrane is suspended in the cell culture container such thatthe first surface is proximal and in spaced relation to a bottom surfaceof the cell culture container, thereby defining within the cell culturecontainer a lower volume comprising the endocardial cells and an uppervolume comprising a second surface of the porous membrane. The shearforce is applied to the second surface of the porous membrane in theupper volume. Optionally, endothelial cells can be plated on the secondsurface of the porous membrane, and the shear force applied upon theplated endothelial cells.

The endocardial cells can comprise smooth muscle cells.

When the vascular pathological condition is atrial fibrillation, oratrial fibrillation and associated hypertension, the cell types cancomprise endothelial cells, smooth muscle cells, endocardial cells, or acombination thereof. Preferably, the cell types are endothelial; smoothmuscle; endothelial and smooth muscle; endocardial; or endocardial andendothelial.

For a vascular pathological condition such as atrial fibrillation, oratrial fibrillation and associated hypertension, the plated cell typescan be from a normal subject, a subject having diabetes, a hypertensivesubject, an aged subject, or an animal genetically modified to modeldiabetes, hypertension, or aging.

When the vascular pathological condition is atrial fibrillation, oratrial fibrillation and associated hypertension, the flow or hemodynamicpattern can be derived from a cardiac sinus or from an atrialfibrillation rhythm.

When the vascular pathological condition is atrial fibrillation, oratrial fibrillation and associated hypertension, the factor can compriseoxLDL, TNFα, aldosterone, angiotensin II, or a combination thereof. Forexample, the factor(s) can comprise oxLDL; TNFα; oxLDL and TNFα;aldosterone; angiotensin II; aldosterone and angiotensin II; oxLDL,TNFα, and angiotensin II; oxLDL, TNFα, and aldosterone; or oxLDL, TNFα,aldosterone, and angiotensin II.

For a vascular pathological condition where a porous membrane is used,smooth muscle cells can be plated on a first surface of the porousmembrane. The porous membrane is suspended in the cell culture containersuch that the first surface is proximal and in spaced relation to abottom surface of the cell culture container, thereby defining withinthe cell culture container a lower volume comprising the smooth musclecells and an upper volume comprising a second surface of the porousmembrane. The shear force is applied to the second surface of the porousmembrane in the upper volume. Optionally, endothelial cells can beplated on the second surface of the porous membrane.

For a vascular pathological condition where a porous membrane is used,endothelial cells can be plated on a second surface of a porousmembrane. The porous membrane is suspended in the cell culture containersuch that a first surface of the porous membrane is proximal and inspaced relation to a bottom surface of the cell culture container,thereby defining within the cell culture container a lower volumecomprising the first surface of the porous membrane and an upper volumecomprising the endothelial cells. The shear force is applied to theendothelial cells in the upper volume. Optionally, smooth muscle cellscan be plated on the first surface of the porous membrane.

When the vascular pathological condition is an advanced inflammation,such as atherosclerosis, the cell types can comprise endothelial cells,smooth muscle cells, or a combination thereof. Preferably, the celltypes are endothelial; smooth muscle; or endothelial and smooth muscle.

When the vascular pathological condition is an advanced inflammation,such as atherosclerosis, the plated cell types can be from a normalsubject, a subject having diabetes, a hypertensive subject, or an animalgenetically modified to model diabetes or hypertension.

When the vascular pathological condition is advanced inflammation, suchas atherosclerosis, the flow or hemodynamic pattern can be atheroprone,atheroprotective (i.e., also described herein as “healthy state”),derived from a femoral artery, or derived from an arteriole.

When the vascular pathological condition is advanced inflammation suchas atherosclerosis, the factor can comprise LDL, oxLDL, TNFα, HDL, atriglyceride-rich lipoprotein, or a combination thereof. For example,the factor(s) can comprise LDL; LDL and oxLDL; oxLDL; HDL; HDL andoxLDL; TNFα; TNFα and oxLDL; TNFα, oxLDL, and HDL; or TNFα, oxLDL, and atriglyceride-rich lipoprotein.

When the vascular pathological condition is an advanced inflammation,such as hypertriglyceridemia, the cell types can comprise endothelialcells, smooth muscle cells, or a combination thereof. Preferably, thecell types are endothelial; smooth muscle; or endothelial and smoothmuscle.

When the vascular pathological condition is an advanced inflammation,such as hypertriglyceridemia, the plated cell types can be from a normalsubject, a subject having diabetes, a hypertensive subject, or an animalgenetically modified to model diabetes or hypertension.

When the vascular pathological condition is advanced inflammation, suchas hypertriglyceridemia, the flow or hemodynamic pattern can beatheroprone, atheroprotective, derived from a femoral artery, or derivedfrom an arteriole.

When the vascular pathological condition is advanced inflammation suchas hypertriglyceridemia, the factor can comprise a triglyceride-richlipoprotein.

When the vascular pathological condition is abdominal aortic aneurysm,the cell types can comprise endothelial cells, smooth muscle cells, or acombination thereof. Preferably, the cell types are endothelial; smoothmuscle; or endothelial and smooth muscle.

When the vascular pathological condition is abdominal aortic aneurysm,the plated cell types can be from a normal subject, a subject havingdiabetes, a hypertensive subject, a smoker, a subject having abdominalaortic aneurysm, or an animal genetically modified to model diabetes orhypertension or modified to model abdominal aortic aneurysm.

When the vascular pathological condition is abdominal aortic aneurysm,the flow or hemodynamic pattern can be derived from an abdominal arteryor derived from an intra-abdominal aortic aneurysm rhythm.

When the vascular pathological condition is abdominal aortic aneurysm,the factor can comprise oxLDL, TNFα, glucose, an elastin degradationproduct, elastase, angiotensin II, aldosterone, insulin, TGF-β, or acombination thereof. For example, the factor(s) can be oxLDL; TNFα;glucose; an elastin degradation product; elastase; angiotensin II;aldosterone; insulin; TGF-β; oxLDL and TNFα; oxLDL and glucose; oxLDLand an elastin degradation product; oxLDL and elastase; oxLDL andangiotensin II; oxLDL and aldosterone; oxLDL and insulin; oxLDL andTGF-β; TNFα and glucose; TNFα and an elastin degradation product; TNFαand elastase; TNFα and angiotensin II; TNFα and aldosterone; TNFα andinsulin; TNFα and TGF-β; glucose and an elastin degradation product;glucose and elastase; glucose and angiotensin II; glucose andaldosterone; glucose and insulin; glucose and TGF-β; an elastindegradation product and elastase; an elastin degradation product andangiotensin II; an elastin degradation product and aldosterone; anelastin degradation product and insulin; an elastin degradation productand TGF-β; elastase and angiotensin II; elastase and aldosterone;elastase and insulin; elastase and TGF-β; angiotensin II andaldosterone; angiotensin II and insulin; angiotensin II and TGF-β;aldosterone and insulin; aldosterone and TGF-β; insulin and TGF-β;oxLDL, TNFα, and glucose; oxLDL, TNFα, and an elastin degradationproduct; oxLDL, TNFα, and elastase; oxLDL, TNFα, and angiotensin II;oxLDL, TNFα, and aldosterone; oxLDL, TNFα, and insulin; oxLDL, TNFα, andTGF-β; TNFα, glucose, and an elastin degradation product; TNFα, glucose,and elastase; TNFα, glucose, and angiotensin II; TNFα, glucose, andaldosterone; TNFα, glucose, and insulin; TNFα, glucose, and TGF-β; andthe like.

When the vascular pathological condition is abdominal aortic aneurysm,smoke extract can be added to the culture media.

When the vascular pathological condition is a diabetic vascularcondition, such as diabetic nephropathy, diabetic neuropathy, ordiabetic retinopathy, the cell types can comprise endothelial cells,smooth muscle cells, or a combination thereof. Preferably, the celltypes are endothelial; smooth muscle; or endothelial and smooth muscle.

When the vascular pathological condition is a diabetic vascularcondition, such as diabetic nephropathy, diabetic neuropathy, ordiabetic retinopathy, the plated cell types can be from a normalsubject, a subject having diabetes, or an animal genetically modified tomodel diabetes.

When the vascular pathological condition is a diabetic vascularcondition, such as diabetic nephropathy, diabetic neuropathy, ordiabetic retinopathy, the flow or hemodynamic pattern can beatheroprone, atheroprotective, derived from a femoral artery, or derivedfrom an arteriole.

When the vascular pathological condition is a diabetic vascularcondition, such as diabetic nephropathy, diabetic neuropathy, ordiabetic retinopathy, the factor can comprise oxLDL, TNFα, glucose, HDL,oxHDL, a triglyceride-rich lipoprotein, insulin, or a combinationthereof. For example, the factor(s) can comprise glucose; glucose andinsulin; glucose, oxLDL, and TNFα; glucose, insulin, oxLDL, and TNFα;glucose, oxLDL, TNFα, and HDL; glucose, oxLDL, TNFα, and oxHDL; glucose,oxLDL, TNFα, HDL, and oxHDL; glucose, insulin, oxLDL, TNFα, and HDL;glucose, insulin, oxLDL, TNFα, and oxHDL; glucose, insulin, oxLDL, TNFα,HDL, and oxHDL; glucose, oxLDL, TNFα, and a triglyceride-richlipoprotein; or glucose, insulin, oxLDL, TNFα, and a triglyceride-richlipoprotein.

When the vascular pathological condition is hypertension, the cell typescan comprise endothelial cells, smooth muscle cells, or a combinationthereof. Preferably, the cell types are endothelial; smooth muscle; orendothelial and smooth muscle.

When the vascular pathological condition is hypertension, the platedcell types can be from a normal subject, a subject having diabetes, ahypertensive subject, or an animal genetically modified to modeldiabetes or hypertension.

When the vascular pathological condition is hypertension, the flow orhemodynamic pattern can be atheroprone, atheroprotective, or derivedfrom a femoral artery, a pulmonary artery, or an arteriole.

When the vascular pathological condition is hypertension, the factor cancomprise oxLDL, TNFα, angiotensin II, aldosterone, or a combinationthereof. For example, the factor(s) can comprise angiotensin II;aldosterone; angiotensin II and aldosterone; or angiotensin II,aldosterone, oxLDL and TNFα.

When the vascular pathological condition is artery calcification, thecell types can comprise endothelial cells, smooth muscle cells, or acombination thereof. Preferably, the cell types are endothelial; smoothmuscle; or endothelial and smooth muscle.

When the vascular pathological condition is artery calcification, theplated cell types can be from a normal subject, a subject havingdiabetes, a hypertensive subject, or an animal genetically modified tomodel diabetes or hypertension.

When the vascular pathological condition is artery calcification, theflow or hemodynamic pattern can be atheroprone, atheroprotective, orderived from a femoral artery, a pulmonary artery, or an arteriole.

When the vascular pathological condition is artery calcification, thefactor can comprise oxLDL, TNFα, vitamin D, an inorganic phosphate,leptin, adiponectin, or a combination thereof. For example, thefactor(s) can comprise oxLDL; TNFα; vitamin D; an inorganic phosphate;leptin; adiponectin; oxLDL and TNFα; oxLDL and vitamin D; oxLDL and aninorganic phosphate; oxLDL and leptin; oxLDL and adiponectin; TNFα andvitamin D; TNFα and an inorganic phosphate; TNFα and leptin; TNFα andadiponectin; vitamin D and an inorganic phosphate; vitamin D and leptin;vitamin D and adiponectin; an inorganic phosphate and leptin; aninorganic phosphate and adiponectin; leptin and adiponectin; oxLDL,TNFα, and vitamin D; oxLDL, TNFα, and an inorganic phosphate; oxLDL,TNFα, and leptin; oxLDL, TNFα, and adiponectin; TNFα, vitamin D, and aninorganic phosphate; TNFα, vitamin D, and leptin; TNFα, vitamin D, andadiponectin; and the like.

When the vascular pathological condition is thrombosis, the cell typescan comprise endothelial cells, smooth muscle cells, or a combinationthereof. Preferably, the cell types are endothelial; smooth muscle; orendothelial and smooth muscle.

When the vascular pathological condition is thrombosis, the plated celltypes can be from a normal subject, a subject having diabetes, ahypertensive subject, or an animal genetically modified to modeldiabetes or hypertension.

When the vascular pathological condition is thrombosis, the flow orhemodynamic pattern can be atheroprone, atheroprotective, or derivedfrom a femoral artery, a pulmonary artery, or an arteriole.

When the vascular pathological condition is thrombosis, the factor cancomprise TNFα, oxLDL, glucose, or a combination thereof. For example,the factor(s) can comprise TNFα; oxLDL; glucose; or oxLDL and glucose.

When the pathological condition is fatty liver disease, the cell typescan comprise hepatocytes, nonparenchymal hepatic cells, or combinationsthereof. The nonparenchymal hepatic cells can include sinusoidalendothelial cells, hepatic stellate cells, Kupffer cells, orcombinations thereof.

When the vascular pathological condition is fatty liver disease, theflow or hemodynamic pattern can be from a normal subject, a subjecthaving fatty liver disease, or an animal genetically modified to modelfatty liver disease.

Where the pathological condition is fatty liver disease and a porousmembrane is used, hepatocytes can be plated on a first surface of theporous membrane. The porous membrane is suspended in the cell culturecontainer such that the first surface is proximal and in spaced relationto a bottom surface of the cell culture container, thereby definingwithin the cell culture container a lower volume comprising thehepatocytes and an upper volume comprising a second surface of theporous membrane. The shear force is applied to the second surface of theporous membrane in the upper volume. Optionally, nonparenchymal hepaticcells can be plated on the second surface of the porous membrane, andthe shear force is applied to the nonparenchymal hepatic cells in theupper volume. Optionally, an extracellular matrix component can bedeposited on the first surface of the porous membrane, and subsequentlyhepatoctyes can be plated on the extracellular matrix component.

Where the pathological condition is fatty liver disease and a porousmembrane is used, nonparenchymal hepatic cells can be plated on a secondsurface of a porous membrane. The porous membrane is suspended in thecell culture container such that a first surface of the porous membraneis proximal and in spaced relation to a bottom surface of the cellculture container, thereby defining within the cell culture container alower volume comprising the first surface of the porous membrane and anupper volume comprising the nonparenchymal hepatic cells. The shearforce is applied to the nonparenchymal hepatic cells in the uppervolume. Optionally, an extracellular matrix component can be depositedon the first surface of the porous membrane, and subsequentlyhepatoctyes can be plated on the extracellular matrix component.

When the vascular pathological condition is fatty liver disease, thefactor can comprise insulin, glucose, or a combination thereof. Forexample, the factor(s) can comprise insulin; glucose; or insulin andglucose.

When the pathological condition is diabetes, the cell type can compriseβ-cells and the factor can comprise insulin, glucose, or insulin andglucose.

Physiologic Conditions

The physiologic conditions that can be mimicked in the methods of theinvention include the physiologic conditions corresponding to anypathological condition of interest, such pathological conditions beingdescribed herein. For example, a physiologic condition corresponding tofatty liver disease can be a healthy liver state, and a physiologiccondition corresponding to atherosclerosis can be an atheroprotectivestate.

Flow Devices

The shear force can be applied using any suitable flow device which iscapable of inducing flow of the culture media, wherein the flow mimicsflow to which the cell type or cell types being cultured are exposed invivo in the pathological or physiological condition. For example, theflow device can be a cone-and-plate device or a parallel plate flowdevice.

The flow device can be a cone-and-plate device substantially asdescribed in U.S. Pat. No. 7,811,782 and in Hastings, et al.,Atherosclerosis-prone hemodynamics differentially regulates endothelialand smooth muscle cell phenotypes and promotes pro-inflammatory priming,AMERICAN J. PHYSIOLOGY & CELL PHYSIOLOGY 293:1824-33 (2007), thecontents of each of which are hereby incorporated by reference. Anexample of such a device is depicted in FIG. 15B. The device 200comprises an electronic controller for receiving a set of electronicinstructions, a motor 220 operated by the electronic controller, and ashear force applicator operatively connected to the motor for beingdriven by the motor. The shear force applicator can comprise a cone 230which is attached to the motor, and the cone can be directly driven bythe motor. The motor causes the cone to rotate in either direction(clockwise or counterclockwise).

The cone-and-plate device accommodates a cell culture container, forexample a Petri dish (e.g., a 75-mm diameter Petri dish). The cone isadapted to fit inside the cell culture container. Thus, for example, ina device adapted for use with 75-mm diameter Petri dishes, the cone hasa diameter of about 71.4 mm. The cone generally has a shallow coneangle. For example, the angle between the surface of the cone and thesurface within the Petri dish is approximately 1°.

When the cone of the device is submerged in culture media in the Petridish and rotated by the motor, the cone exerts a rotational force uponthe culture media, and this in turn applies shear force to cells platedwithin the cell culture container or to a surface of a porous membranesuspended in the cell culture container.

The cone-and-plate device can also include a base for securely holdingthe cell culture container. The device can also include clips that mounton the Petri dish and secure inflow and outflow tubing which is used toperfuse the upper and lower volumes, as described further below.

The flow can be derived from a previously measured hemodynamic pattern,and can be modeled into a set of electronic instructions. The shearforce is based on the set of electronic instructions. The flow devicecomprises an electronic controller for receiving the set of electronicinstructions. The motor is operated by the electronic controller. Ashear force applicator operatively connected to the motor is driven bythe motor. Preferably, the shear force applicator comprises a coneattached to the motor.

The flow device is used in conjunction with a cell culture container.The cell culture container can include inlets and outlets for the flowof cell culture media, factors, drugs, compounds and other componentsinto and out of the cell culture container.

The inlets and outlets for the flow device can be secured to the cellculture container by a clip. FIG. 23 depicts such a clip. Each clip ismade up of three parts: the main body 1 and two pieces of thin metaltubing 2 and 3 as shown in FIG. 23. The clip can be secured to the sideof a cell culture dish from the outside by a screw 4. For example, twoclips can be attached and tightened to the side of the dish from theoutside by a screw 4, as shown in FIGS. 24A and B). The main body 1 ismade of treated stainless steel metal and angles around the edge of thedish for attachment and access purposes. Two pieces of thin metal tubing(2 and 3) per clip are bent to provide access to the dish for supplyingand drawing off media efficiently, without obstructing the conerotation. A set screw 5 on either side of the main body 1 secures themetal tubing 2, 3 to the main body and holds the metal tubing in placesuch that it extends to the correct depth within the culture media.Flexible tubing then slides over the metal tubing, which is used to drawmedia (e.g., from the source bottle to the dish via mechanicalperistaltic pump in the device of the examples).

FIGS. 24A and 24B show the clips positioned in a cell culture container.In the configurations shown in FIGS. 24A and 24B, a porous membranesuspended is suspended in the cell culture container, with endothelialcells only (FIG. 24B) or endothelial cells and smooth muscle cells (FIG.24A) plated on surfaces of the porous membrane.

Hemodynamic Patterns

The hemodynamic pattern can be derived from a subject or subjects havingthe pathological condition or a disease-promoting condition. Thedisease-promoting condition can comprise atrophy, calculi, choristoma,pathologic constriction, pathologic dilation, diverticulum, hypertrophy,polyps, prolapse, rupture, an arteriovenous fistula, or an appendage(e.g., a left atrial appendage).

The hemodynamic pattern can be derived from at least a portion of anartery, an arteriole, a vein, a venule, or an organ.

When a hemodynamic pattern is derived from at least a portion of anartery or an arteriole, the artery or arteriole can comprise a carotidartery, thoracic artery, abdominal artery, pulmonary artery, femoralartery, renal efferent artery, renal afferent artery, coronary artery,brachial artery, internal mammary artery, cerebral artery, aorta,pre-capillary arteriole, hepatic artery, anterior cerebral artery,middle cerebral artery, posterior cerebral artery, basilar artery,external carotid artery, internal carotid artery, vertebral artery,subclavian artery, aortic arch, axillary artery, internal thoracicartery, branchial artery, deep branchial artery, radial recurrentartery, superior epigastric artery, descending aorta, inferiorepigastric artery, interosseous artery, radial artery, ulnar artery,palmar carpal arch, dorsal carpal arch, superficial or deep palmar arch,digital artery, descending branch of the femoral circumflex artery,descending genicular artery, superior genicular artery, inferiorgenicular artery, anterior tibial artery, posterior tibial artery,peroneal artery, deep plantar arch, arcuate artery, common carotidartery, intercostal arteries, left or right gastric artery, celiactrunk, splenic artery, common hepatic artery, superior mesentericartery, renal artery, inferior mesenteric artery, testicularis artery,common iliac artery, internal iliac artery, external iliac artery,femoral circumflex artery, perforating branch, deep femoral artery,popliteal artery, dorsal metatarsal artery, or dorsal digital artery.

When a hemodynamic pattern is derived from at least a portion of an veinor venule, the vein or venule can comprise a post-capillary venule,saphenous vein, hepatic portal vein, superior vena cava, inferior venacava, coronary vein, Thesbian vein, superficial vein, perforator vein,systemic vein, pulmonary vein, jugular vein, sigmoid sinus, externaljugular vein, internal jugular vein, inferior thyroid vein, subclavianvein, internal thoracic vein, axillary vein, cephalic vein, branchialvein, intercostal vein, basilic vein, median cubital vein,thoracoepigastric vein, ulnar vein, median antebranchial vein, inferiorepigastric vein, deep palmar arch, superficial palmar arch, palmardigital vein, cardiac vein, inferior vena cava, hepatic vein, renalvein, abdominal vena cava, testicularis vein, common iliac vein,perforating branch, external iliac vein, internal iliac vein, externalpudendal vein, deep femoral vein, great saphenous vein, femoral vein,accessory saphenous vein, superior genicular vein, popliteal vein,inferior genicular vein, great saphenous vein, small saphenous vein,anterior or posterior tibial vein, deep plantar vein, dorsal venousarch, or dorsal digital vein.

When a hemodynamic pattern is derived from at least a portion of anorgan, the organ can comprise a liver, a kidney, a lung, a brain, apancreas, a spleen, a large intestine, a small intestine, a heart, askeletal muscle, an eye, a tongue, a reproductive organ, or an umbilicalcord.

The hemodynamic pattern can be derived from analysis of ultrasound data.

The hemodynamic pattern can be derived from analysis of magneticresonance imaging (MRI) data.

The flow or the hemodynamic pattern can be time-variant.

The flow or the hemodynamic pattern can be derived from a chamber of theheart, a left atrial appendage during sinus rhythm, an atrialfibrillation, or a ventricular fibrillation.

When the flow or the hemodynamic pattern is derived from a chamber ofthe heart, the chamber of the heart can comprise a left atrium, a rightatrium, a left ventricle or a right ventricle.

The flow or the hemodynamic pattern can result from a physical changeresulting from a pathological condition.

The flow or hemodynamic pattern can be derived from a subject whereinblood flow or a hemodynamic pattern has been altered as a direct orindirect effect of administration of a drug to a subject as compared tothe flow or the hemodynamic pattern for the subject absentadministration of the drug.

The flow or the hemodynamic pattern can be derived from an animal, suchas a genetically modified animal or a human Preferably, the pattern isderived from a human

Cell Types

Cell types for use in methods of the invention include primary cells andimmortalized cells. The primary cells or immortalized cells can comprisecells isolated from at least one subject having the pathological orphysiologic condition, cells isolated from at least one subject having arisk factor for the pathological condition, cells isolated from at leastone subject with a single nucleotide polymorphism linked to apathological condition, cells isolated from at least one subject with anidentified genotype linked to drug toxicity, or cells isolated from atleast one subject with a single nucleotide polymorphism linked to drugtoxicity.

The primary cells or the immortalized cells used in in vitro methods ofthe invention involving a physiologic condition comprise cells isolatedfrom at least one subject having the physiologic condition, cellsisolated from at least one subject having a risk factor for apathological condition, cells isolated from at least one subject with asingle nucleotide polymorphism linked to a pathological condition, cellsisolated from at least one subject with an identified genotype linked todrug toxicity, or cells isolated from at least one subject with a singlenucleotide polymorphism linked to drug toxicity.

The primary cells or immortalized cells used in in vitro methods of theinvention involving a pathological condition can comprise cells isolatedfrom at least one subject having the pathological condition, cellsisolated from at least one subject having a risk factor for thepathological condition, cells isolated from at least one subject with asingle nucleotide polymorphism linked to the pathological condition,cells isolated from at least one subject with an identified genotypelinked to drug toxicity, or cells isolated from at least one subjectwith a single nucleotide polymorphism linked to drug toxicity.

The primary cells or immortalized cells used in in vitro methods of theinvention involving a pathological condition can comprise cells isolatedfrom at least one subject not having the pathological condition, cellsisolated from at least one subject not having a risk factor for thepathological condition, cells isolated from at least one subject withouta single nucleotide polymorphism linked to the pathological condition,cells isolated from at least one subject without an identified genotypelinked to drug toxicity, or cells isolated from at least one subjectwithout a single nucleotide polymorphism linked to drug toxicity.

The primary cells or immortalized cells used in in vitro methods of theinvention involving a pathological condition can comprise cells isolatedfrom at least one subject having a different pathological condition,cells isolated from at least one subject having a risk factor for adifferent pathological condition, or cells isolated from at least onesubject with a single nucleotide polymorphism linked to a differentpathological condition.

When the cells are isolated from at least one subject having a riskfactor for the pathological condition, the risk factor can include, butis not limited to, smoking, age, gender, race, epigenetic imprinting, anidentified genotype linked to the pathological condition, an identifiedsingle nucleotide polymorphism linked to the pathological condition,diabetes, hypertension, atherosclerosis, atherosclerotic plaque rupture,atherosclerotic plaque erosion, thoracic aortic aneurysm, cerebralaneurysm, abdominal aortic aneurysm, cerebral aneurysm, heart failure,stroke, Marfan syndrome, carotid intima-medial thickening, atrialfibrillation, kidney disease, pulmonary fibrosis, chronic obstructivepulmonary disease, pulmonary artery disease, pulmonary hypertension,hyperlipidemia, familial hypercholesterolemia, peripheral arterydisease, deep vein thrombosis, vascular restenosis, vascularcalcification, myocardial infarction, obesity, hypertriglyceridemia,hypoalphalipoproteinemia, fatty liver disease, hepatitis C, hepatitis B,liver fibrosis, bacterial infection, viral infection, cirrhosis, liverfibrosis, or alcohol-induced liver disease.

The primary cells can include a cell lineage derived from stem cells(e.g., adult stem cells, embryonic stem cells, inducible pluripotentstem cells, or bone marrow-derived stem cells) or stem-like cells. Thecell lineage derived from stem cells or stem-like cells can compriseendothelial cells, smooth muscle cells, cardiac myocytes, hepatocytes,neuronal cells, or endocrine cells.

Cell types for use in methods of the invention include renal cells,cells of the airways, blood-brain barrier cells, vascular cells, hepaticcells, pancreatic cells, cardiac cells, muscle cells, spleen cells,gastrointestinal tract cells, skin cells, liver cells, immune cells, orhematopoietic cells.

Specific cell types for use in the methods include astrocytes,endothelial cells, glomerular fenestrated endothelial cells, renalepithelial podocytes, alpha cells, β-cells, delta cells, pancreaticpolypeptide (PP) cells, epsilon cells, glial cells, hepatocytes,neurons, nonparenchymal hepatic cells, podocytes, smooth muscle cells,mesangial cells, pericytes, cardiac muscle cells, skeletal muscle cells,leukocytes, monocytes, myocytes, macrophages, neutrophils, dendriticcells, T-cells, B-cells, endothelial progenitor cells, stem cells,circulating stem cells, and circulating hematopoietic cells. Thenonparenchymal hepatic cells include hepatic stellate cells, sinusoidalendothelial cells, and Kupffer cells. Preferably, the specific celltypes can include endothelial cells, smooth muscle cells, hepatocytes,sinusoidal endothelial cells, or a combination thereof.

The cell types for use in the methods of the invention can be animalcell types, such as cells from a genetically modified animal. The animalcell types are preferably human cell types. The human cell types can beselected on the basis of age, gender, race, epigenetics, disease,nationality, the presence or absence of one or more single nucleotidepolymorphisms, a risk factor as described herein, or some othercharacteristic that is relevant to the pathological or physiologiccondition.

The shear force applied in the methods of the invention can be appliedindirectly to the at least one plated cell type.

The shear force applied in the methods of the invention can be applieddirectly to the at least one plated cell type.

The cell types, additional components such as extracellular matrixcomponent, and the porous membrane are within the culture media (i.e.,covered with culture media) in the methods of the invention.

The methods of the invention can further comprise analyzing at least oneof the cell types for toxicity, inflammation, permeability,compatibility, cellular adhesion, cellular remodeling, cellularmigration, or phenotypic modulation resulting from the drug or thecompound.

Cell Culture Media

Standard cell culture media can be used in the methods of the invention.

Factors Added to Cell Culture Media

The factors that can be added to the cell culture media are describedthroughout the specification in conjunction with an associatedpathological or physiologic condition.

In Vivo Factor Concentrations

The physiologic in vivo concentrations of the factors are well known inthe art, as are the methods of determining these in vivo concentrations.For example, the respective in vivo concentrations of HDL in a healthyhuman and in a human having atherosclerosis are greater than 30 mg/dl to200 mg/dl, and less than 30 mg/dl, as determined from whole blood.Methods for determining in vivo concentrations of factors are availablein the United States Pharmacopeia and in other literature.

A reported in vivo concentration range for a factor can vary dependingupon the method used for determining the range, the source from whichthe factor is obtained (e.g., whole blood or serum), the medicalcondition of the patient (i.e., whether the patient has a pathologicalcondition or physiologic condition), and time of day relative to normalsleep and eating schedule. However, it would be known to one of ordinaryskill in the art that a concentration outside an in vivo physiologicalconcentration range reported in the literature would be an in vivopathological concentration using the method reported for determining theconcentration. Likewise, a concentration below the lower endpoint orabove the upper endpoint of an in vivo pathological concentration rangereported in the literature would be an in vivo physiologic concentrationusing the method reported for determining the concentration; whether thein vivo physiologic concentration is below the lower endpoint or abovethe upper endpoint will depend upon the factor. For example, the in vivophysiologic concentration of the factor HDL would be above the upperendpoint of the range, but the in vivo physiologic concentration of thefactor oxLDL would be below the lower endpoint of the range, as would berecognized by one of ordinary skill in the art.

Other Components

Extracellular Matrix Components

Extracellular matrix components for use in the methods of the inventioncan comprise heparan sulfate, chondroitin sulfate, keratan sulfate,hyaluronic acid, a collagen, an elastin, a fibronectin, a laminin, avitronectin, or combinations thereof. Collagen is a preferredextracellular matrix component, and is preferably the type of collagenthat is present in the in vivo environment of the cell type or celltype(s) that are plated for a particular pathological or physiologiccondition.

The extracellular matrix component can be secreted by fibroblasts,chondrocytes, or osteoblasts plated on the surface within the cellculture container.

The extracellular matrix component is especially suitable for use in themethods of the invention involving the liver.

Drug or Compound

The drug or compound can be an anti-inflammatory agent, ananti-neoplastic agent, an anti-diabetic agent, a protein kinaseinhibitor, an anti-thrombotic agent, a thrombolytic agent, ananti-platelet agent, an anti-coagulant, a calcium channel blocker, achelating agent, a rho kinase inhibitor, an anti-hyperlipidemic agent,an agent that raises HDL, an anti-restenosis agent, an antibiotic, animmunosuppressant, an anti-hypertensive agent, a diuretic, an anorectic,an appetite suppressant, an anti-depressant, an anti-psychotic, acontraceptive, a calcimimetic, a biologic medical product, or acombination thereof.

When the drug is an anti-inflammatory agent, the anti-inflammatory agentcan comprise a steroid (e.g., prednisone, hydrocortisone, prednisolone,betamethasone, or dexamethasone), a non-steroidal anti-inflammatory drug(NSAID) (e.g., a salicylate, ibuprofen, acetaminophen, naproxen, orketoprofen), a selective cyclooxygenase inhibitor (e.g., celecoxib orrofecoxib), a non-selective cyclooxygenase inhibitor, an immuneselective anti-inflammatory agent (e.g., phenylalanine-glutamine-glycinetripeptide), or a combination thereof.

When the drug comprises an anti-neoplastic agent, the anti-neoplasticagent can comprise an alkylating agent (e.g., cisplatin, carboplatin,oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucide, orifosfamide), an anti-metabolite (e.g., azathioprine or mercaptopurine),a plant alkaloid (e.g., a taxane such as paclitaxel or docetaxel, avinca alkaloid such as vincristine, vinblastine, or vindesine, or apodophyllotoxin such as etoposide or teniposide), a topoisomeraseinhibitor (e.g., irinotecan, topotecan, or amsacrine), a cytotoxicantibiotic (e.g., actinomycin, bleomycin, plicamysin, mitomycin,doxorubicin, daunorubicin, valrubicin, idarubicin, or epirubicin), or acombination thereof.

When the drug is an anti-diabetic agent, the anti-diabetic agent cancomprise a biguanide (e.g., metformin), a thiazolidinedione (e.g.,rosiglitazone, troglitazone, or pioglitazone), a sulfonylurea (e.g.,tolbutamine, acetohexamide, tolazamide, chlorpropamide, glipazide,glyburide, glimepiride, gliclazide, glycopyramide, or gliquidone), anincretin mimetic (e.g., exenatide, liraglutide, or taspoglutide), adipeptidyl peptidase IV inhibitor (e.g., vildagliptin, sitagliptin,saxaglitpin, linagliptin, alogliptin, or septagliptin), a sodium-glucoseco-transporter 2 inhibitor (e.g., dapagliflozin, canagliflozin,empagliflozin, ipragliflozin, remogliflozin, or sergliflozin), or aglucokinase activator (e.g., piragliatin).

When the drug comprises a protein kinase inhibitor, the protein kinaseinhibitor can comprise a serine/threonine-specific kinase inhibitor, atyrosine-specific kinase inhibitor (e.g., imatinib, bevacizumab,cetuximab, axitinib, lapatinib, ruxolitinib, or sorafenib), an epidermalgrowth factor (EGF) receptor inhibitor, a fibroblast growth factor (FGF)receptor inhibitor, a platelet-derived growth factor (PDGF) receptorinhibitor, or a vascular endothelial growth factor (VEGF) receptorinhibitor.

When the drug comprises the anti-thrombotic agent, the anti-thromboticagent can comprise dipyridamole, urokinase, r-urokinase, r-prourokinase,reteplase, alteplase, streptokinase, rt-PA, TNK-rt-PA, monteplase,staphylokinase, pamiteplase, unfractionated heparin, or APSAC.

When the drug comprises the thrombolytic agent, the thrombolytic agentcan comprise a streptokinase, a urokinase, or a tissue plasminogenactivator.

When the drug comprises the anti-platelet agent, the anti-platelet agentcan comprise a glycoprotein IIb/IIIa inhibitor, a thromboxane inhibitor,an adenosine diphosphate receptor inhibitor, a prostaglandin analogue,or a phosphodiesterase inhibitor. For example, the anti-platelet agentcan comprise clopidogrel, abciximab, tirofiban, orbofiban, xemilofiban,sibrafiban, roxifiban, or ticlopinin.

When the drug comprises the anti-coagulant, the anti-coagulant cancomprise a vitamin K antagonist (e.g., warfarin), a factor Xa inhibitor(e.g., apixaban, betrixaban, edoxaban, otamixaban, rivaroxaban,fondaparinux, or idraparinux), or a direct thrombin inhibitor (e.g.,hirudin, bivalirudin, lepirudin, desirudin, dabigatran, ximelagatran,melagatran, or argatroban).

When the drug comprises the calcium channel blocker, the calcium channelblocker can comprise verapamil, diltiazem, amlodipine, aranidipine,azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine,isradipine, efonidipine, felodipine, lacidipine, lercanidipine,manidipine, nicardipine, nifedipine, nilvadipine, nimodipine,nisoldipine, nitrendipine, or pranidipine.

When the drug comprises the chelating agent, the chelating agent cancomprise penicillamine, triethylene tetramine dihydrochloride, EDTA,DMSA, deferoxamine mesylate, or batimastat.

When the drug comprises the rho kinase inhibitor, the rho kinaseinhibitor can comprise Y27632.

When the drug comprises the anti-hyperlipidemic agent, theanti-hyperlipidemic agent can comprise a statin (e.g., atorvastatin,cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,pravastatin, rosuvastatin, or simvastatin), a fibrate (e.g.,bezafibrate, ciprofibrate, clofibrate, gemfibrozil, or fenofibrate), aselective inhibitor of dietary cholesterol absorption (e.g., ezetimibe),or a cholesterylester transfer protein inhibitor (e.g., anacetrapib,dalcetrapib, torcetrapib, or evacetrapib).

When the drug comprises the agent that raises HDL, the agent that raisesHDL can comprise an inhibitor of proprotein convertase subtilisin/kexintype 9 (PCSK9), such as AMG145.

When the drug comprises the anti-restenosis agent, the anti-restenosisagent can comprise dexamethasone ticlopidine, clopidogrel, sirolimus,paclitaxel, zotarolimus, everolimus, or umirolimus.

When the drug comprises the antibiotic, the antibiotic can compriseactinomycin-D.

When the drug comprises the immunosuppressant, the immunosuppressant cancomprise a glucocorticoid, methotrexate, azathioprine, mercaptopurine,dactinomycin, mitomycin C, bleomycin, mithramycin, ciclosporin,tacrolimus, sirolimus, an interferon, infliximab, etanercept, oradalimumab.

When the drug comprises the anti-hypertensive agent, theanti-hypertensive agent can comprise a beta adrenergic receptorantagonist (e.g., alprenolol, bucindolol, carteolol, carvedilol,labetalol, nadolol, oxprenolol, penbutalol, pindolol, propranolol,sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol,metoprolol, or nebivolol), an angiotensin II receptor antagonist (e.g.,losartan, olmesartan, valsartan, telmisartan, irbesartan, orazilsartan), or an angiotensin converting enzyme inhibitor (e.g.,captopril, enalapril, lisinopril, quinapril, zofenopril, imidapril,benazepril, trandolapril, or ramipril).

When the drug comprises the diuretic, the diuretic can comprisefuroseamide, amiloride, spironolactone, or hydrochlorothiazide.

When the drug comprises the anorectic, the anorectic can comprisephentermine, fenfluramine, dexfenfluramine, sibutramine, lorcaserin,topiramate, or a combination thereof.

When the drug comprises the anti-depressant, the anti-depressant cancomprise imipramine, desipramine, amitryptiline, paroxetine, citalopram,fluoxetine, or escitalopram.

When the drug comprises the anti-psychotic, the anti-psychotic cancomprise aripiprazole, risperidone, olanzapine, quetiapine, cariprazine,lurasidone, or asenapine.

When the drug comprises the contraceptive, the contraceptive cancomprise a combination of drospirenone and ethinyl estradiol.

When the drug comprises the calcimimetic, the calcimimetic can comprisecinacalcet.

When the drug comprises the biologic medical product, the biologicmedical product can comprise a synthetic polysaccharide, a synthetic,partially synthetic or humanized immunoglobulin, or a recombinanttherapeutic protein.

The drug or the compound can comprise a radiocontrast agent, aradio-isotope, a prodrug, an antibody fragment, an antibody, a livecell, a therapeutic drug delivery microsphere, microbead, nanoparticle,gel or cell-impregnated gel, or a combination thereof.

The compound can be capable of inhibiting, activating, or altering thefunction of proteins or genes in the at least one cell type.

When the drug or the compound is to be evaluated for elution from avascular stent material, the method can further comprise testing atleast one of the cell types for compatibility with, cellular adhesionto, or phenotypic modulation by the vascular stent material. Thevascular stent material can be adjacent to the endothelial cells or thesmooth muscle cells.

Effect on the Physiologic or Pathological Condition

In methods of testing a drug or a compound for an effect, the effect cancomprise an effect on a physiologic condition or an effect on apathological condition. For example, the effect on the physiologiccondition or the pathological condition can be a toxic effect, aprotective effect, a pathologic effect, a disease-promoting effect, aninflammatory effect, an oxidative effect, an endoplasmic reticulumstress effect, a mitochondrial stress effect, an apoptotic effect, anecrotic effect, a remodeling effect, a proliferative effect, an effecton the activity of a protein, such as inhibition of a protein oractivation of a protein, or an effect on the expression of a gene, suchas an increase in the expression of the gene or a decrease in theexpression of the gene.

Multiple Cell Type Configurations for the Flow Device

The methods of the invention can further comprise perfusing culturemedia, factors, drugs or compounds into and out of the cell container.

When the surface within the cell culture container comprises a porousmembrane suspended in the cell culture container, the method can furtherinclude the step of plating at least one cell type on a surface withinthe cell culture container comprising plating a first cell type on afirst surface of a porous membrane, and optionally plating a second celltype on a second surface of the porous membrane, wherein the porousmembrane is suspended in the cell culture container such that the firstsurface is proximal and in spaced relation to a bottom surface of thecell culture container, thereby defining within the cell culturecontainer a lower volume comprising the first cell type and an uppervolume comprising the optional second cell type. The porous membrane canbe adapted to permit fluid communication of the cell culture media andphysical interaction and communication between cells of the first celltype and cells of the optional second cell type. The shear force isapplied to the second cell type or the second surface of the porousmembrane in the upper volume. The method can further comprise perfusingculture media into and out of the upper volume and perfusing culturemedia into and out of the lower volume. The method can further compriseperfusing a drug or the compound into at least one of the upper volumeand the lower volume.

When the surface within the cell culture container comprises a porousmembrane suspended in the cell culture container, the method can furtherinclude the step of plating at least one cell type on a surface withinthe cell culture container comprising optionally plating a first celltype on a first surface of a porous membrane, and plating a second celltype on a second surface of the porous membrane, wherein the porousmembrane is suspended in the cell culture container such that the firstsurface is proximal and in spaced relation to a bottom surface of thecell culture container, thereby defining within the cell culturecontainer a lower volume comprising the optional first cell type and anupper volume comprising the second cell type. The porous membrane can beadapted to permit fluid communication of the cell culture media andphysical interaction and communication between cells of the optionalfirst cell type and cells of the second cell type. The shear force isapplied to the second cell type in the upper volume. The method canfurther comprise perfusing culture media into and out of the uppervolume and perfusing culture media into and out of the lower volume. Themethod can further comprise perfusing a drug or the compound into atleast one of the upper volume and the lower volume.

The inlets and outlets in the cell culture container can be within theportions of the cell culture container defining the upper and lowervolumes.

The methods described in this section can further comprise analyzing atleast one of the first cell type or the second cell type for toxicity,inflammation, permeability, compatibility, cellular adhesion, cellularremodeling, cellular migration, or phenotypic modulation resulting fromthe drug or the compound.

These methods can further comprise plating a third cell type on asurface of the container or the first surface or second surface of theporous membrane, suspending a third cell type in the culture mediawithin the upper volume, or suspending a third cell type in the culturemedia within the lower volume.

These methods can further comprise plating a fourth cell type on asurface of the container or the first or second surface of the porousmembrane, suspending a fourth cell type in the culture media within theupper volume, or suspending a fourth cell type in the culture mediawithin the lower volume.

These methods can further comprise plating a fifth cell type on asurface of the container or the first or second surface of the porousmembrane, suspending a fifth cell type in the culture media within theupper volume, or suspending a fifth cell type in the culture mediawithin the lower volume.

The first, second, third, fourth and fifth cell types can be variousprimary or immortalized cell types as described in the section aboveregarding cell types.

In each of these combinations, the cells of the third cell type, thecells of the fourth cell type or the cells of the fifth cell type can beadhered to the bottom surface of the container.

Definitions

For purposes of the inventions described herein, the term“disease-promoting condition” means an abnormal anatomical condition(i.e., the anatomy of the vasculature that deviates significantly from amedically accepted normal anatomy) that can contribute to a diseasestate.

The term “factor” means a biological substance that contributes to theproduction of a pathological or physiologic condition. Preferably, thefactor provides a change in a level of a marker of the pathological orphysiologic condition in the at least one plated cell type or in theculture media upon application of the shear force, as compared to thelevel of the marker in the at least one plated cell type or in theculture media in the absence of application of the shear force.

The term “hemodynamic” means blood flow that mimics the blood flow invivo in a tissue of interest. For example, when arterial blood flow isof interest, the acceleration/deceleration rates, flow reversal, forwardbasal flow, etc. are some parameters characterizing arterial hemodynamicflow. In other tissues, such as the liver, a constant blood flow may beused to characterize in vivo hemodynamics.

The term “pathological condition” means an abnormal anatomical orphysiological condition, which includes the objective or subjectivemanifestation of a disease.

The term “physiologic condition” means a normal medical state that isnot pathologic, and can be a medical state characteristic of orconforming to the normal functioning or state of the body or a tissue ororgan.

The term “subject” means an animal (e.g., a genetically modified animalor a human) The animal can include a mouse, rat, rabbit, cat, dog, orprimate, or any animal typically used in medical research.

The use of the methods of the invention for particular in vitro modelsis described below.

Thrombosis

The present methods can be used to model thrombosis in vitro. In thecoagulation cascade, thrombin converts fibrinogen to fibrin, which isdeposited on the surface of a blood vessel to begin blood clot formation(thrombosis). TNFα is a potent inflammatory cytokine. TNFα and othercytokines have been shown to be potent mediators of endothelial andsmooth muscle cell-derived tissue factor in vitro, which mediates fibrindeposition in the vascular wall. Circulating levels of TNFα detected inhumans with cardiovascular disease are about 0.01 ng/ml to about 0.1ng/ml. In healthy individuals, circulating levels of TNFα are much loweror undetectable, for example about 0 ng/ml to about 0.001 ng/ml.

In the methods which model thrombosis in vitro, endothelial cells areplated on a surface within a cell culture container. The surface withinthe cell culture container can be the surface of a porous membrane, andthe porous membrane can be suspended in the cell culture container suchthat a first surface of the porous membrane is proximal and in spacedrelation to a bottom surface of the cell culture container, therebydefining within the cell culture media a lower volume comprising thefirst surface of the porous membrane and an upper volume comprising thesecond surface of the porous membrane and the endothelial cells.Alternatively, the surface upon which the endothelial cells are platedis the bottom of the cell culture container.

One or more additional cell types can be plated on a surface within thecell culture container or suspended in the media in the cell culturecontainer. For example, smooth muscle cells can be plated on a firstsurface of a porous membrane within the cell culture container andendothelial cells can be plated on a second surface of the porousmembrane. The porous membrane is suspended in the cell culture containersuch that the first surface of the porous membrane is proximal and inspaced relation to a bottom surface of the cell culture container,thereby defining within the cell culture media a lower volume comprisingthe first surface of the porous membrane and the smooth muscle cells andan upper volume comprising the second surface of the porous membrane andthe endothelial cells.

Monocytes, macrophages, neutrophils, endothelial progenitor cells,circulating stem cells, circulating hematopoietic cells, or leukocytescan optionally be suspended in the cell culture media within the upperor lower volume.

A shear force is applied upon the plated endothelial cells, the shearforce resulting from the flow of the culture media induced by ahemodynamic flow device. The flow mimics the flow to which endothelialcells are exposed in vivo at regions of the vasculature where thrombosisis likely to occur. For example, the flow is atheroprone hemodynamicflow.

The shear force can be applied upon the plated endothelial cells for aperiod of time prior to the addition of one or more factors to theculture media. For example, shear force may be applied to theendothelial cells for a period of about 12 hours to about 48 hours,about 12 hours to about 36 hours, about 16 hours to about 32 hours, orabout 18 hours to about 28 hours prior to the addition of one or morefactors to the culture media. For instance, the shear force can beapplied to the plated endothelial cells for about 24 hours prior to theaddition of one or more factors. Alternatively, the shear force can beapplied upon the plated endothelial cells concurrently with the additionof the one or more factors to the culture media.

One or more factors can be added to the culture media. For example, theone or more factors added to the culture media can be factors which areinvolved in the development or progression of thrombosis. The factor orfactors are added to the media in a concentration that is within an invivo concentration range of the factor observed in subjects withvascular disease. For example, TNFα can be added to the culture media ina concentration that is within the in vivo concentration range for TNFαwhich is observed in individuals with vascular disease. For example,TNFα can be added to the culture media in a concentration of about 0.005ng/ml to about 0.2 ng/ml, about 0.01 ng/ml to about 0.1 ng/ml, about0.03 ng/ml to about 0.07 ng/ml, or about 0.04 ng/ml to about 0.06 ng/ml.TNFα can be added to the culture media at a concentration of about 0.05ng/ml or about 0.1 ng/ml.

Other factors can also be added to the culture media in addition to theTNFα. For example, oxidized LDL (oxLDL), glucose, or both oxLDL andglucose can be added the culture media in combination with TNFα. Suchfactors are added to the culture media in concentrations which arewithin the in vivo concentration ranges of the factors observed insubjects with vascular disease. In healthy individuals, plasmaconcentrations of oxLDL are generally less than about 25 μg/ml, while inpatients with vascular disease, the plasma concentration of oxLDL isgreater than about 25 μg to about 100 μg/ml. Thus, for example, oxLDLcan be added to the culture media in a concentration of about 25 μg/mlto about 120 μg/ml, about 30 μg/ml to about 100 μg/ml, about 40 μg/ml toabout 80 μg/ml, or about 25 μg/ml to about 50 μg/ml. For instance, oxLDLcan be added to the culture media in a concentration of about 25 μg/mlor about 50 μg/ml.

Glucose can also be added to the culture media. Diabetes and theassociated elevated glucose levels are risk factors for thrombosis. Inhealthy individuals, blood glucose concentrations are about 5 mM toabout 10 mM, while in diabetic individuals, blood glucose concentrationsrange from greater than about 10 mM to about 20 mM. Thus, for example,glucose can be added to the culture media in a concentration of about 10mM to about 25 mM, about 12 mM to about 20 mM, or about 14 mM to about18 mM. For instance, glucose can be added to the culture media in anamount of about 15 mM or about 17.5 mM.

Application of the shear stress to the plated endothelial cells issuitably continued for a period of time following the addition of theone or more factors to the cell culture media.

Application of the shear stress can be continued, for example, for aperiod of about 12 hours to about 48 hours, about 18 hours to about 36hours, or about 20 to about 30 hours, about 18 hours to about 72 hours,or about 24 hours to about 72 hours. For instance, the shear stress canbe continued for about 24 hours following the addition of the one ormore factors to the cell culture media.

Clot formation can then induced by incubating the endothelial cells withplatelet-free plasma (PLP), calcium, and fibrinogen. This incubation canbe performed under static conditions. Alternatively, the shear forceapplication to the endothelial cells can be continued during thisincubation. The cell culture media can be removed from the upper volumeand the endothelial cells can subsequently be incubated with the PLP,calcium, and fibrinogen, with or without continued application of shearto the endothelial cells. Alternatively, the PLP, calcium, andfibrinogen can be added to the cell culture media in the upper volume,with or without the continued application of shear forces.

Mimicking of thrombosis can be assessed by any of a number of methods.In general, a change in a level of a marker of thrombosis in theendothelial cells or smooth muscle cells or in the culture media uponapplication of the shear force, as compared to the level of the markerin the endothelial cells or smooth muscle cells or in the culture mediain the absence of application of the shear force, confirms mimicking ofthrombosis. For example, mimicking of thrombosis can be assessed byexamining fibrin deposition, by examining the expression of genes orproteins and/or secreted microparticles or proteins relevant tothrombosis, or by examining the activity of proteins relevant tothrombosis.

Atherosclerosis

The present methods can also be used to model atherosclerosis in vitro.Atherosclerosis is a focal inflammatory disease marked by inflammatorysignaling within regions of the vasculature where low and oscillatingshear stresses (atheroprone shear stresses) “prime” the endothelium foran inflammatory response. An important mediator of the inflammatoryresponse is the transcription factor NFκB, which is activated byatheroprone shear stresses in vivo and in vitro. Oxidized low-densitylipoprotein (oxLDL) is a hallmark of advanced atherosclerosis, is foundin atherosclerotic lesions, and is elevated in the circulation ofpatients with cardiovascular complications. Oxidized1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), a majorcomponent of oxLDL, has not been shown to act through the canonical NFκBpathway, and although oxLDL is capable of increasing the expression ofNFκB-dependent genes in vitro in static monocultures, this oftenrequires higher concentrations of oxLDL (>100 μg/ml) than those observedin vivo in individuals with cardiovascular disease. In healthy patientsplasma concentrations of oxLDL are on average 7 μg/ml, while in patientswith myocardial infarction average plasma concentrations are about 28 toabout 34 μg/ml, with some patients having levels of about 60 μg/ml. TNFαis also secreted in advanced atherosclerotic lesions and is elevated inthe circulation of patients who have experienced myocardial infarction.TNFα is a potent, pro-inflammatory cytokine capable of activating NFκBsignaling at high concentrations (>1 ng/ml).

The previous in vitro studies were all performed within staticmonocultures of endothelial cells. In the present methods, by contrast,atheroprone hemodynamic shear forces “prime” monocultures of endothelialcells or co-cultures of endothelial cells and smooth muscle cells byactivating NFκB signaling, and the addition of oxLDL and/or TNFα, andoptionally certain other factors at concentrations which are within thein vivo concentration range of the factor which is observed in patientswith vascular disease further enhances NFκB activity and downstreaminflammatory signaling.

In the present methods which model atherosclerosis in vitro, endothelialcells are plated on a surface within a cell culture container. Thesurface within the cell culture container can be the surface of a porousmembrane, and the porous membrane can be suspended in the cell culturecontainer such that a first surface of the porous membrane is proximaland in spaced relation to a bottom surface of the cell culturecontainer, thereby defining within the cell culture media a lower volumecomprising the first surface of the porous membrane and an upper volumecomprising the second surface of the porous membrane and the endothelialcells. Alternatively, the surface upon which the endothelial cells areplated is the bottom of the cell culture container.

One or more additional cell types can be plated on a surface within thecell culture container or suspended in the media in the cell culturecontainer. For example, smooth muscle cells can be plated on a firstsurface of a porous membrane within the cell culture container andendothelial cells can be plated on a second surface of the porousmembrane. The porous membrane is suspended in the cell culture containersuch that the first surface of the porous membrane is proximal and inspaced relation to a bottom surface of the cell culture container,thereby defining within the cell culture media a lower volume comprisingthe first surface of the porous membrane and the smooth muscle cells andan upper volume comprising the second surface of the porous membrane andthe endothelial cells.

Monocytes, macrophages, neutrophils, endothelial progenitor cells,circulating stem cells, circulating hematopoietic cells, or leukocytescan optionally be suspended in the cell culture media within the upperor lower volume.

A shear force is applied upon the plated endothelial cells, the shearforce resulting from the flow of the culture media induced by ahemodynamic flow device. The flow mimics the flow to which endothelialcells are exposed in vivo at regions of the vasculature whereatherosclerosis is likely to occur. For example, the flow is atheropronehemodynamic flow.

The shear force can be applied upon the plated endothelial cells for aperiod of time prior to the addition of one or more factors to theculture media. For example, shear force can be applied to theendothelial cells for a period of about 12 hours to about 48 hours,about 12 hours to about 36 hours, about 16 hours to about 32 hours, orabout 18 hours to about 28 hours prior to the addition of one or morefactors to the culture media. For instance, the shear force can beapplied to the plated endothelial cells for about 24 hours prior to theaddition of one or more factors. Alternatively, the shear force can beapplied upon the plated endothelial cells concurrently with the additionof the one or more factors to the culture media.

One or more factors can be added to the culture media. For example, theone or more factors added to the culture media can be factors which areinvolved in the development or progression of atherosclerosis. Suchfactor or factors are added to the media in a concentration which iswithin the in vivo concentration range of the factor observed in theindividuals with vascular disease.

oxLDL can be added to the culture media in a concentration that iswithin the in vivo concentration range of oxLDL observed in theindividuals with vascular disease. Thus, for example, oxLDL can be addedto the culture media in a concentration of about 25 μg/ml to about 120μg/ml, about 30 μg/ml to about 100 μg/ml, about 40 μg/ml to about 80μg/ml, or about 25 μg/ml to about 50 μg/ml. For instance, oxLDL can beadded to the culture media in a concentration of about 25 μg/ml or about50 μg/ml.

Other factors can also be added to the culture media, either instead ofor in combination with oxLDL. These factors include, but are not limitedto: TNFα, high-density lipoprotein (HDL); triglycerides;triglyceride-rich lipoproteins including very low-density lipoprotein(vLDL), vLDL remnants, chylomicrons, and/or chylomicron remnants;low-density lipoprotein (LDL); glucose; insulin; a fatty acid; TGFβ orcombinations thereof. For example, TNFα can be added to the mediainstead of oxLDL. Alternatively, both oxLDL and TNFα can be added to themedia. HDL can also optionally be added to the media. For example, HDLcan be added to the media alone, or in combination with other factorssuch as TNFα and oxLDL. Triglycerides or triglyceride rich lipoproteinsincluding vLDL, vLDL remnants, chylomicrons, and/or chylomicron remnantscan also optionally added to the media. Glucose can also optionally beadded to the media. For example, glucose may be added to the mediaalone, or in combination with other factors such as TNFα. LDL or TGFβcan also be added to the media.

The factors are added to the media in concentrations which are withinthe in vivo concentration range of the factor observed in theindividuals with vascular disease. Thus, for example, TNFα can be addedto the culture media in a concentration of about 0.005 ng/ml to about0.2 ng/ml, about 0.01 ng/ml to about 0.1 ng/ml, about 0.03 ng/ml toabout 0.07 ng/ml, or about 0.04 ng/ml to about 0.06 ng/ml. For example,TNF a can be added to the culture media in a concentration of about 0.05ng/ml or about 0.1 ng/ml.

oxLDL can be added to the media in a concentration of about 50 μg/ml.

TNFα can be added to the culture media at a concentration of about 0.05ng/ml.

HDL can be added to the culture media in a concentration that is withinan in vivo concentration range of HDL observed in individuals withvascular disease or in individuals at risk for vascular disease. HDLconcentrations in individuals at risk for vascular disease are generallyless than about 300 μg/ml, while HDL concentrations in healthyindividuals range from greater than about 300 μg/ml up to about 2,000μg/ml in healthy exercising patients. Thus, for example, HDL can beadded to the culture media in a concentration of about 1 μg/ml to about300 μg/ml, about 10 μg/ml to about 250 μg/ml, about 45 μg/ml to about200 μg/ml, or about 90 μg/ml to about 150 μg/ml. For example, HDL can beadded to the culture media at a concentration of about 45 μg/ml or about90 μg/ml.

HDL may suitably be added to the culture media in combination with TNFαand oxLDL. The HDL, TNFα, and oxLDL are suitably each present at aconcentration that is within the in vivo concentration ranges for thesefactors which are observed in individuals with vascular disease, forexample, the concentration ranges listed above for each of thesecomponents. For example, HDL is added to the culture media in aconcentration of 45 μg/ml or 90 μg/ml, TNFα is added to the culturemedia in a concentration of about 0.05 ng/ml, and oxLDL is added to theculture media in a concentration of about 50 μg/ml.

Triglycerides or triglyceride-rich lipoproteins including verylow-density lipoprotein (vLDL), vLDL remnants, chylomicrons, and/orchylomicron remnants can be added to the culture media in concentrationsthat are within the in vivo concentration ranges for these factors whichare observed in individuals with vascular disease. Triglyceride levelsin healthy patients range from about 40 mg/dL to about 150 mg/dL. Inpatients with hypertriglyceridemia, triglyceride levels range fromgreater than about 200 mg/dL to about 1500 mg/dL. Thus, for exampletriglycerides are suitably added to the culture media in a concentrationof about 175 mg/dL to about 1600 mg/dL, about 200 mg/dL to about 1500mg/dL, about 400 mg/dL to about 1200 mg/dL, or about 600 mg/dL to about1000 mg/dL.

Diabetes and the associated elevated levels of blood glucose are riskfactors for atherosclerosis. Therefore, glucose may also suitably beadded to the media in the present methods for modeling atherosclerosisin vitro. The glucose is added to the culture media at a concentrationthat is within the in vivo concentration range for glucose as observedin individuals with diabetes. In healthy individuals, blood glucoseconcentrations are about 5 to about 10 mM, while in diabeticindividuals, blood glucose concentrations range from greater than about10 mM to about 20 mM. Thus, for example, glucose is suitably added tothe culture media in a concentration of about 10 mM to about 25 mM,about 12 mM to about 20 mM, or about 14 mM to about 18 mM. For instance,glucose can be added to the culture media in an amount of about 15 mM orabout 17.5 mM.

Glucose can be added to the culture media together with TNFα. Theglucose and TNFα are added to the culture media in concentrations thatare within the in vivo concentration ranges for glucose and TNFα whichare observed in individuals with diabetes or vascular disease forexample, the concentration ranges listed above for each of thesecomponents. For example, glucose is suitably added to the media at aconcentration of about 15 mM and TNFα is suitably added to the culturemedia at a concentration of 0.05 ng/ml.

When both glucose and TNFα are added to the culture media, the glucosecan be added to the culture media and the cells cultured in the presenceof the glucose for a period of time prior to the application of theshear stress. For example, the cells are suitably cultured in thepresence of the glucose for about 1 to about 7 days, for example about 3to about 5 days, or about 4 days prior to the application of shearstress. Shear stress can then applied to the upon the plated endothelialcells for a period of time prior to the addition of the TNFα to theculture media. For example, shear stress can be applied to theendothelial cells for a period of about 12 hours to about 48 hours,about 12 hours to about 36 hours, about 16 hours to about 32 hours,about 18 hours to about 28 hours, or about 24 hours prior to theaddition of the TNFα to the culture media.

LDL or TGFβ can added to the media at concentrations that are within anin vivo concentration range of LDL or TGFβ which is observed inindividuals with vascular disease. In healthy individuals, LDL levelsgenerally range from about 50 mg/dL to about 100 mg/dL, while inindividuals with atherosclerosis, LDL levels are generally above about100 mg/dL. Thus, for example, LDL is suitably added to the culture mediaat a concentration of about 100 mg/dL to about 500 mg/dL, about 100mg/dL to about 300 mg/dL, or about 100 mg/dL.

TGFβ levels in healthy individuals are generally less than about 30ng/ml, while levels in individuals with vascular disease are about 30ng/ml to about 100 ng/ml. Thus, TGFβ is suitably added to the culturemedia in a concentration of about 30 ng/ml to about 150 ng/ml, about 30ng/ml to about 100 ng/ml, about 50 to about 100 ng/ml, or about 60 toabout 90 ng/ml.

Application of the shear stress to the plated endothelial cells issuitably continued for a period of time following the addition of theone or more factors to the cell culture media. Application of the shearstress can be continued, for example, for a period of about 12 hours toabout 48 hours, about 18 hours to about 36 hours, or about 20 to about30 hours, about 18 hours to about 72 hours, or about 24 hours to about72 hours. For instance, the shear stress can be continued for about 24hours following the addition of the one or more factors to the cellculture media.

Mimicking of atherosclerosis can be assessed by a of a number ofmethods. In general, a change in a level of a marker of atherosclerosisin the endothelial cells or smooth muscle cells or in the culture mediaupon application of the shear force, as compared to the level of themarker in the endothelial cells or smooth muscle cells or in the culturemedia in the absence of application of the shear force confirmsmimicking of atherosclerosis. For example, mimicking of atherosclerosiscan be assessed by examining the expression of genes or proteinsrelevant to atherosclerosis, by examining the activity of proteinsrelevant to atherosclerosis, or by examining levels of secretedcytokines.

Hypertension

The methods of the present invention can also be used to modelhypertension in vitro. Angiotensin II (ANG2) levels are increased inpatients with cardiovascular complications, such as atherosclerosis,diabetes or hypertension. Typical concentrations of ANG2 range fromabout 1 nM to about 5 nM in healthy patients, and from greater thanabout 6 nM to about 20 nM in hypertensive patients. In addition,aldosterone is an important signaling hormone downstream of ANG2 in therenin-angiotensin system. Its levels can vary under a number ofpathologies, including atherosclerosis, diabetes, and hypertension.Concentrations of aldosterone in healthy individuals are about 0.3 mM.Concentrations of aldosterone in individuals with hyperaldosteronismrange from about 0.8 mM to about 1 mM.

In the methods which model hypertension in vitro, endothelial cells areplated on a surface within a cell culture container. The surface withinthe cell culture container can be the surface of a porous membrane, andthe porous membrane can be suspended in the cell culture container suchthat a first surface of the porous membrane is proximal and in spacedrelation to a bottom surface of the cell culture container, therebydefining within the cell culture media a lower volume comprising thefirst surface of the porous membrane and an upper volume comprising thesecond surface of the porous membrane and the endothelial cells.Alternatively, the surface upon which the endothelial cells are platedcan be the bottom of the cell culture container.

One or more additional cell types can be plated on a surface within thecell culture container or suspended in the media in the cell culturecontainer. For example, smooth muscle cells can be plated on a firstsurface of a porous membrane within the cell culture container andendothelial cells can be plated on a second surface of the porousmembrane. The porous membrane is suspended in the cell culture containersuch that the first surface of the porous membrane is proximal and inspaced relation to a bottom surface of the cell culture container,thereby defining within the cell culture media a lower volume comprisingthe first surface of the porous membrane and the smooth muscle cells andan upper volume comprising the second surface of the porous membrane andthe endothelial cells.

Monocytes, macrophages, neutrophils, endothelial progenitor cells,circulating stem cells, circulating hematopoietic cells, or leukocytescan optionally be suspended in the cell culture media in the upper orlower volume.

A shear force is applied upon the plated endothelial cells, the shearforce resulting from the flow of the culture media induced by ahemodynamic flow device. The flow mimics the flow to which endothelialcells are exposed in vivo in hypertension.

The shear force can be applied upon the plated endothelial cells for aperiod of time prior to the addition of one or more factors to theculture media. For example, shear force can be applied to theendothelial cells for a period of about 12 hours to about 48 hours,about 12 hours to about 36 hours, about 16 hours to about 32 hours, orabout 18 hours to about 28 hours prior to the addition of one or morefactors to the culture media. For instance, the shear force is appliedto the plated endothelial cells for about 24 hours prior to the additionof one or more factors. Alternatively, the shear force is applied uponthe plated endothelial cells concurrently with the addition of the oneor more factors to the culture media.

One or more factors can be added to the culture media. For example, theone or more factors added to the culture media can be factors which areinvolved in the development or progression of hypertension. The factoror factors are added to the media in a concentration that is within anin vivo concentration range of the factor observed in subjects withvascular disease. For example, angiotensin is suitably added to theculture media at a concentration of about 5.5 nM to about 25 nM, about 6nM to about 20 nM, about 8 nM to about 15 nM, or about 9 nM to about 12nM, e.g., a concentration of 10 nM.

The angiotensin may be added to the culture media either alone or incombination with another factor such as aldosterone. Alternatively,aldosterone can be added to the culture media by itself or incombination with factors other than angiotensin. When aldosterone isadded to culture media, it is suitably present at a concentration ofabout 0.5 mM to about 1.5 mM, or about 0.8 mM to about 1 mM, e.g., at aconcentration of about 1 mM.

Application of the shear stress to the plated endothelial cells issuitably continued for a period of time following the addition of theone or more factors to the cell culture media. Application of the shearstress can be continued, for example, for a period of about 12 hours toabout 48 hours, about 18 hours to about 36 hours, or about 20 to about30 hours, about 18 hours to about 72 hours, or about 24 hours to about72 hours. For example, the shear stress can be continued for about 24hours following the addition of the one or more factors to the cellculture media.

Mimicking of atherosclerosis can be assessed by a number of methods. Ingeneral, a change in a level of a marker of atherosclerosis in theendothelial cells or smooth muscle cells or in the culture media uponapplication of the shear force, as compared to the level of the markerin the endothelial cells or smooth muscle cells or in the culture mediain the absence of application of the shear force confirms mimicking ofatherosclerosis. For example, mimicking of atherosclerosis can beassessed by examining the expression of genes or proteins and/orsecreted microparticles or proteins relevant to atherosclerosis, byexamining the activity of proteins relevant to atherosclerosis, or byexamining levels of secreted cytokines, chemokines, or growth factors.

Physiologic Liver Model

The present methods can also be used to create a physiologic in vitromodel of the liver. In such methods, hepatocytes are plated on a surfacewithin a cell culture container, and shear forces are applied indirectlyto the plated hepatocytes. For example, the hepatocytes are suitablyplated on a first surface of a porous membrane, where the porousmembrane is suspended in a cell culture container such that the firstsurface is proximal and in spaced relation to a bottom surface of thecell culture container, thereby defining within the cell culturecontainer a lower volume comprising the hepatocytes and an upper volumecomprising a second surface of the porous membrane. The shear force isapplied to the second surface of the porous membrane in the upper volumeof the container. Thus, the configuration of cells in the device (FIG.15C) is based on in vivo microarchitecture of hepatic lobules (FIG.15A).

As shown in FIG. 15A, in hepatic lobules in vivo, cords of hepatocytes100 are separated from sinusoidal blood flow 150 by a filtering layer ofsinusoidal endothelial cells 110 and a layer of extracellular matrix140. The layer of extracellular matrix 140 provides for anchorage of thehepatocytes, is involved in signaling, and provides a reservoir ofcytokines and growth factors. The hepatocytes 110 have a polarizedmorphology and biliary canaliculi 120 are present in the hepatocytelayer. Sinusoidal blood flow 150 and interstitial blood flow 130 providefor oxygen and nutrient transport.

FIGS. 15B and 15C depict an exemplary configuration used in the presentin vitro liver model. As shown in the inset in FIG. 15B and in FIG. 15C,hepatocytes 260 are plated on a porous membrane 250 suspended in a cellculture container 240, and a shear force applicator (shown as a cone 230in FIGS. 15B and 15C) is used to apply a shear force upon the opposingside of the porous membrane. The shear force results from the flow ofculture media in the cell culture container. The porous membrane actsanalogously to the filtering layer of sinusoidal endothelial cells whichis present in the liver. The hepatocytes are shielded from directeffects of flow, as they would be in vivo. Inlets and outlets 270 in theupper and lower volumes within the cell culture container allow for thecontinuous perfusion of culture media and for perfusion of drugs orcompounds into and out of the cell culture media. Application of theshear force creates controlled hemodynamics that regulate interstitialflow and solute transfer through the porous membrane. In the in vitromodels of the present invention, the hepatocytes maintain theirpolarized morphology and bile canaliculi.

As illustrated in FIG. 15C, at least one layer of one or moreextracellular matrix components 280 (e.g., a collagen gel) can suitablybe deposited on a first surface of the porous membrane. The hepatocytes260 are then plated on the extracellular matrix component(s). One ormore additional layers of the extracellular matrix component(s) can thenbe deposited on top of the hepatocytes, such that the hepatocytes aresubstantially surrounded by the extracellular matrix component(s). Theextracellular matrix component suitably comprises heparan sulfate,chondroitin sulfate, keratan sulfate, hyaluronic acid, a collagen, anelastin, a fibronectin, a laminin, a vitronectin, or combinationsthereof. For example, the extracellular matrix component can comprisecollagen.

One or more additional cell types can be plated on a surface within thecell culture container or suspended in the culture media. For example,nonparenchymal hepatic cells are suitably plated on the second surfaceof the porous membrane, and the shear force is applied to the platednon-parenchymal cells. The nonparenchymal cells may include hepaticstellate cells, sinusoidal endothelial cells, Kupffer cells, orcombinations thereof. The hepatocytes and nonparenchymal hepatic cellsare suitably primary cells isolated from the liver of an animal, forexample from the liver of a human. Alternatively, the hepatocytes and/orthe nonparenchymal hepatic cells are immortalized cells.

Media is suitably continuously perfused on both sides of the porousmembrane, while shear forces, derived from a range of physiologicalblood flow values, are continuously applied to the second surface of theporous membrane or to the plated nonparenchymal hepatic cells. The shearforces applied to the second surface of the porous membrane mimic theflow through hepatic sinusoids which occurs in vivo. The shear rate issuitably about 0.1 dynes/cm² to about 3.0 dynes/cm², about 0.2 dynes/cm²to about 2.5 dynes/cm², about 0.3 dynes/cm² to about 1.0 dynes/cm² orabout 0.4 dynes/cm² to about 0.8 dynes/cm². For example, the shear ratecan be about 0.6 dynes/cm². Alternatively, the shear rate can be about2.0 dynes/cm².

In the physiologic in vitro liver model, one or more factors are presentin the culture media. These one or more factors can be added to themedia at concentrations which are capable of maintaining the mimickingof the physiologic liver condition in vitro for a period of time underthe shear force, where the same concentrations of these factors areincapable of maintaining the mimicking of the physiologic livercondition in vitro for the period of time in the absence of the shearforce. For example, the factors may comprise insulin, glucose, or acombination of insulin and glucose. The glucose and insulin are suitablypresent in reduced concentrations as compared to the concentrationswhich are typically used in static cultures (about 17.5 mM glucose andabout 2 μM insulin). For example, the glucose may be present in theculture media at a concentration of about 5 mM to about 10 mM, or at aconcentration of about 5.5 to about 7 mM, e.g., at a concentration ofabout 5.5 mM. The insulin may be present in the culture media at aconcentration of about 0.05 nM to about 5 nM, for example about 0.1 nMto about 3 nM, or about 0.5 to about 2.5 nM, e.g., at a concentration ofabout 2 nM. The one or more factors are suitably added to the culturemedia before or concurrently with application of the shear force.

The concentrations of the one or more factors are suitably capable ofmaintaining the mimicking of the physiologic liver condition in vitrofor at least about 7 days, at least about 14, days, at least about 21days, at least about 30 days, or longer.

Mimicking of the physiologic liver condition can be assessed by a numberof methods. In general, a change in a level of a marker of thephysiologic liver condition in the hepatocytes or nonparenchymal hepaticcells or in the culture media upon application of the shear force, ascompared to the level of the marker in the hepatocytes or nonparenchymalhepatic cells or in the culture media in the absence of application ofthe shear force confirms mimicking of the physiologic liver condition.For example, mimicking of the physiologic liver condition can beassessed by examining the hepatocytes or nonparenchymal hepatic cellsfor the expression of genes or proteins involved in maintaining theliver in a physiologic state (e.g., in hepatocytes, metabolic andinsulin/glucose/lipid pathway genes); examining the hepatocytes forlipid accumulation; examining the hepatocytes or nonparenchymal hepaticcells for changes in differentiated function (e.g., in hepatocytes,measuring urea and albumin secretion); examining the hepatocytes ornonparenchymal hepatic cells for changes in metabolic activity (e.g., inhepatocytes, using cytochrome p450 assays) or transporter activity; orby examining the hepatocytes or nonparenchymal hepatic cells formorphological changes. The physiologic condition of the liver can alsobe assessed by comparing the response of the hepatocytes ornonparenchymal hepatic cells to xenobiotics, nutrients, growth factorsor cytokines to the in vivo liver response to the same xenobiotics,nutrients, growth factors or cytokines.

Fatty Liver

The methods described herein can also be used to create an in vitromodel of fatty liver disease. Lipid regulation within hepatocytes is acomplex and dynamic process. Triglyceride buildup can occur as aconsequence of increased fatty acid uptake from a high fat diet,increased peripheral lipolysis, or from increased de novo lipogenesis.Insulin and glucose are key regulators of de novo lipogenesis andcontribute to increased triglyceride content within hepatocytes bystimulating triglyceride synthesis as well as inhibiting fatty acidmetabolism by beta oxidation.

Non-alcoholic fatty liver disease (NAFLD) is correlated with obesity,type II diabetes, and metabolic syndrome in the presence of insulinresistance. NAFLD is characterized by hepatic steatosis (excessive lipidaccumulation in the liver) that if left untreated progresses toinflammatory changes (steatohepatitis) and cirrhosis. Many animal modelsinduce steatosis through a hyperglycemic-hyperinsulinemic environment(e.g., through use of a low fat/high carbohydrate diet to stimulatelipogenesis). However, current in vitro hepatocyte models lack anadequate insulin-glucose response to induce the same, probably onaccount of the superphysiological levels of insulin/glucose required tomaintain hepatocytes in culture under static conditions. Such in vitromodels fail to induce fatty changes in hepatocytes through insulin andglucose, perhaps due to impaired insulin responsiveness of hepatocytesunder static culture conditions and rapid dedifferentiation of thehepatocytes in vitro.

By contrast, as described above with respect to the physiological livermodel, hepatocytes cultured in the presence of controlled liver-derivedhemodynamics and transport retain differentiated function, morphology,and response at physiological glucose and insulin levels. In thissystem, introducing high concentrations of insulin and glucose (a“disease milieu”) induces fatty changes in the hepatocytes. Thus,controlled hemodynamics and transport produces a more physiologicalresponse to insulin and glucose in the hepatocytes, thereby inducing thefatty changes associated with steatosis in a hyperinsulemic,hyperglycemic environment as is typically seen initially under insulinresistant conditions of diabetes. In addition, hepatocytes cultured inthe presence of controlled hemodynamics and transport display inductionand toxicity responses to drugs at concentrations much closer to in vivoand clinical C_(max) levels than static culture systems. The presentsystem therefore provides an in vitro model of fatty liver disease.

In this model, the hepatocytes are generally plated in the same manneras described above for the physiological liver model. Hepatocytes areplated on a surface within a cell culture container, and shear forcesare applied indirectly to the plated hepatocytes. For example, thehepatocytes are suitably plated on a first surface of a porous membrane,where the porous membrane is suspended in a cell culture container suchthat the first surface is proximal and in spaced relation to a bottomsurface of the cell culture container, thereby defining within the cellculture container a lower volume comprising the hepatocytes and an uppervolume comprising a second surface of the porous membrane. The shearforce is applied to the second surface of the porous membrane in theupper volume of the container.

At least one layer of one or more extracellular matrix components cansuitably be deposited on the first surface of the porous membrane. Thehepatocytes are then plated on the extracellular matrix component(s).One or more additional layers of the extracellular matrix component(s)can then be deposited on top of the hepatocytes, such that thehepatocytes are substantially surrounded by the extracellular matrixcomponent(s). The extracellular matrix component suitably comprisesheparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid,a collagen, an elastin, a fibronectin, a laminin, a vitronectin, orcombinations thereof. For example, the extracellular matrix componentcan comprise collagen.

One or more additional cell types can be plated on a surface within thecell culture container or suspended in the culture media. For example,nonparenchymal hepatic cells are suitably plated on the second surfaceof the porous membrane, and the shear force is applied to the platednon-parenchymal cells. The nonparenchymal cells may include hepaticstellate cells, sinusoidal endothelial cells, Kupffer cells, orcombinations thereof. The hepatocytes and nonparenchymal hepatic cellsare suitably primary cells isolated from the liver of an animal, forexample from the liver of a human. Alternatively, the hepatocytes and/orthe nonparenchymal hepatic cells are immortalized cells.

Media is suitably continuously perfused on both sides of the porousmembrane, while shear forces, derived from a range of physiologicalblood flow values, are continuously applied to the second surface of theporous membrane or to the plated nonparenchymal hepatic cells. The shearforces applied to the second surface of the porous membrane mimic theflow through hepatic sinusoids which occurs in vivo. The shear rate issuitably about 0.1 dynes/cm² to about 3.0 dynes/cm², about 0.2 dynes/cm²to about 2.5 dynes/cm², about 0.3 dynes/cm² to about 1.0 dynes/cm² orabout 0.4 dynes/cm² to about 0.8 dynes/cm². For example, the shear ratecan be about 0.6 dynes/cm². Alternatively, the shear rate can be about2.0 dynes/cm².

In the in vitro fatty liver model, one or more factors are present inthe culture media. These one or more factors are added to the media atconcentrations which are capable of maintaining the mimicking of fattyliver disease in vitro for a period of time under the shear force, thesame concentration of factor being incapable of maintaining themimicking of fatty liver disease for the period of time in the absenceof the shear force. The factors may comprise, for example, insulin,glucose, or a combination thereof. The glucose is suitably present inthe culture media at a concentration of about 10 mM to about 25 mM,about 12 mM to about 20 mM, or about 14 mM to about 18 mM, e.g., about17.5 mM. The insulin is suitably present in the culture medium at aconcentration of about 1 μM to about 3 μM, about 1.5 μM to about 2.5 μM,or about 1.8 μM to about 2.2 μM, e.g., about 2 μM. The one or morefactors are suitably added to the culture media before or concurrentlywith application of the shear force.

The concentrations of the one or more factors are suitably capable ofmaintaining the mimicking of fatty liver disease condition in vitro forat least about 7 days, at least about 14, days, at least about 21 days,at least about 30 days, or longer.

Mimicking of fatty liver disease can be assessed by a number of methods.In general, a change in a level of a marker of fatty liver disease inthe hepatocytes or nonparenchymal hepatic cells or in the culture mediaupon application of the shear force, as compared to the level of themarker in the hepatocytes or nonparenchymal hepatic cells or in theculture media in the absence of application of the shear force confirmsmimicking of fatty liver disease. For example, mimicking of fatty liverdisease can be assessed by examining the hepatocytes or nonparenchymalhepatic cells for the expression of genes or proteins involved in thefatty liver disease state (e.g., in hepatocytes, metabolic andinsulin/glucose/lipid pathway genes); examining the hepatocytes forlipid accumulation (e.g., in hepatocytes, measuring triglyceride levelsor visualizing lipid droplets); examining the hepatocytes ornonparenchymal hepatic cells for changes in differentiated function(e.g., in hepatocytes, measuring urea and albumin secretion); examiningthe hepatocytes or nonparenchymal hepatic cells for changes in metabolicactivity (e.g., in hepatocytes, using cytochrome p450 assays) ortransporter activity; or by examining the hepatocytes or nonparenchymalhepatic cells for morphological changes. Sequelae to fatty liver changescan also be assessed by measuring the changes in oxidative state of thehepatocytes and the changes in surrounding extracellular matrixcomposition and amount.

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing from thescope of the invention defined in the appended claims.

EXAMPLES Example 1: An In Vitro Model for Arterial and Venous Thrombosis

In the coagulation cascade, thrombin converts fibrinogen to fibrin,which is deposited on the surface of a blood vessel to begin blood clotformation (thrombosis). TNFα is a potent inflammatory cytokine. TNFα andother cytokines have been shown to be potent mediators of endothelialand smooth muscle cell-derived tissue factor in vitro, which mediatesfibrin deposition in the vascular wall. Circulating levels of TNFαdetected in humans with cardiovascular disease are about 0.01 ng/ml toabout 0.1 ng/ml. In healthy individuals, circulating levels of TNFα aremuch lower or undetectable, for example about 0 ng/ml to about 0.001ng/ml.

Methods:

Human endothelial cells were co-cultured with or without smooth musclecells in the presence or absence of human-derived, region-specifichemodynamics. Endothelial cells were exposed to TNFα at variousconcentrations and incubated in human, platelet-free plasma supplementedby ALEXA FLUOR 488 (A488, a fluorescent dye)-labeled fibrinogen.Conversion of A488-fibrinogen to A488-fibrin and deposition on theendothelium was quantified by confocal microscopy.

(i) Static Monoculture Thrombosis Assay

For static monocultures of endothelial cells, endothelial cells wereplated at 100,000 cells/cm² on coverslips and allowed to adhere for 24hours. After 24 hours, media was exchanged with media containing 0ng/ml, 1 ng/ml, 10 ng/ml, or 20 ng/ml TNFα. Cells were incubated for 4hours at 37° C. Following incubation, media was removed and cells werewashed twice with PBS. Cells were then incubated an additional 15minutes at 37° C. with Human Platelet Free Plasma (PFP) supplementedwith 37.5 μg/mL ALEXA-488 human fibrinogen, 20 μg/mL corn trypsininhibitor, and 10 mM calcium. This protocol is depicted in FIG. 1A.After 15 minutes, the cells were fixed with 4% paraformaldehyde (PFA)and stained with 0.5 nM SYT083 (a fluorescent nucleic acid stain) in 10mM Tris, 1 mM EDTA buffer for 45 minutes. Coverslips were mounted ontocoverglass using FLUOROMOUNT-G (a mounting agent) and imaged.

(ii) Monoculture Thrombosis Assay with Shear Stress

Endothelial cells were plated at a plating density of 100,000 cells/cm²on the porous membrane of a TRANSWELL (polycarbonate, 10 μm thicknessand 0.4 μm pore diameter, no. 3419, Corning), and subjected toatheroprone or atheroprotective hemodynamic patterns using acone-and-plate device.

Following 24 hours of shear stress application (atheroprone oratheroprotective), the media was supplemented with TNFα at aconcentration of 0.05 ng/mL or 0.10 ng/mL and the shear stress wascontinued for an additional 24 hours, as shown in FIG. 3A. Media wasthen removed from both the upper and lower chambers, and endothelialcells were washed twice with PBS. Endothelial cells were then incubatedfor 15 minutes at 37° C. with PFP supplemented with 37.5 μg/mL ALEXA-488human fibrinogen, 20 μg/mL corn trypsin inhibitor, and 10 mM calcium.After 15 minutes, the cells were fixed with 4% PFA and stained with 0.5nM SYT083 in 10 mM Tris, 1 mM EDTA buffer for 45 minutes. Small portionsof the porous membrane were mounted onto coverglass using FLUOROMOUNT-Gand imaged.

(iii) Coculture Thrombosis Assays with Shear Stress—Protocol A

Smooth muscle cells were plated on a first surface of the porousmembrane of a TRANSWELL at a plating density of 20,000 cells/cm² andallowed to adhere to the membrane for two hours. The TRANSWELL was theninverted and the cells were incubated in reduced serum growth media(M199 supplemented with 2% FBS, 2 mM L-glutamine, and 100 U/mlpenicillin-streptomycin) for forty-eight hours. Endothelial cells werethen plated on a second surface of the TRANSWELL porous membrane at adensity of 100,000 cells/cm², under the same media conditions andincubated for an additional twenty-four hours prior to the applicationof shear stress.

Following 24 hours of shear stress application (atheroprone oratheroprotective), the media was supplemented with TNFα at aconcentration of 0.05 ng/mL or 0.10 ng/mL and shear stress was continuedfor an additional 24 hours, as shown in FIG. 3A. Media was then removedfrom both the upper and lower chambers, and both the endothelial cellsand smooth muscle cells are washed twice with PBS. Endothelial cellswere then incubated for 15 minutes at 37° C. with PFP supplemented with37.5 μg/mL ALEXA-488 human fibrinogen, 20 μg/mL corn trypsin inhibitor,and 10 mM calcium, and fixed and imaged as described above formonocultures.

(iv) Coculture Thrombosis Assays with Shear Stress—Protocol B

Smooth muscle cells and endothelial cells were plated, subjected toatheroprone or atheroprotective shear stress, and treated with TNFα asdescribed above for Protocol A. PFP supplemented with 37.5 μg/mLALEXA-488 human fibrinogen and 20 μg/mL corn trypsin inhibitor was addedto the upper volume to create a final concentration of PFP ofapproximately 27%. The PFP was further supplemented with calcium for afinal concentration of 10 mM calcium in the PFP/media combination.Endothelial cells were then incubated for an additional 15 minutes at37° C. and fixed and imaged as described above.

(v) Coculture Thrombosis Assays with Shear Stress—Protocol C

Smooth muscle cells and endothelial cells were plated, subjected toatheroprone or atheroprotective shear stress, and treated with TNFα asdescribed above for Protocol A. The cone of the cone-and-plate devicewas then raised by about 2 mm and the in-flow/out-flow clips wereremoved. The cone was then lowered back to the operating height. PFPsupplemented with 37.5 μg/mL ALEXA-488 human fibrinogen, 20 μg/mL corntrypsin inhibitor, was added to the upper volume to create a finalconcentration of PFP of approximately 27%. The PFP was furthersupplemented with calcium for a final concentration of 10 mM calcium inthe PFP/media combination. The co-culture was then incubated for anadditional 30 minutes with application of atheroprone oratheroprotective shear stress, as shown in FIG. 4A. After 30 minutes,the cell culture dish was removed from the device and media was removedfrom the lower volume. The endothelial cells were then fixed and imagedas described above.

Results:

(i) Static Endothelial Cell Monocultures

In cultures of endothelial cells cultured for 24 hours under staticconditions and treated with TNFα for 4 hours, samples treated with 1ng/ml TNFα demonstrated minimal fibrin deposition, whereas samplestreated with 10 ng/ml or 20 ng/ml exhibited dense fibrin networks(1.17e5 vs. 2.69e7 vs. 3.61e7 mean fluorescence intensity, respectively)(FIG. 1B; insets are color images). A cross-section of the clot with the“x-axis” going from left to right and the “z-axis” going up and down wasimaged in a stack as shown in the lower middle panel of FIG. 1B, tomeasure the height of the clot above the surface (z-axis). The mean greyvalue is the fluorescence intensity of each image of the stack at thedesignated height above the cell surface (lower far right panel of FIG.1B).

Fibrin deposition was tissue factor-dependent and blocked by ananti-CD142 antibody. The upper panels of FIG. 1C show fibrin depositionin the presence of an antibody to tissue factor (anti-CD142, left) or acontrol antibody (IgG1K, right). The lower panels show staining ofnuclei in the same fields, demonstrating the presence of cells.

Thus, under static conditions, endothelial cells require activation byTNFα to initiate the clotting cascade. This activation is dependent ontissue factor activity and TNFα concentrations that are approximately200-fold higher than physiological levels.

(ii) Endothelial Cell/Smooth Muscle Cell Co-Cultures Subjected to Shear

FIG. 2B depicts a heat map of relative gene expression in endothelialand smooth muscle cells (grown under atheroprone or atheroprotectiveconditions; see FIG. 2A) of several genes relevant to thrombosis. Therelative changes in gene expression of Tissue Factor (F3) andThrombomodulin (THBD) are presented below the heat map. Atheropronehemodynamics up-regulate Tissue Factor (F3) compared to atheroprotectiveshear stress. Further, thrombomodulin, which binds thrombin and inhibitsthe clotting cascade, is down-regulated. In addition, TNFα stimulationreduces Tissue Factor Pathway Inhibitor (TFPI) in both endothelial andsmooth muscle cells.

Endothelial/smooth muscle cell co-cultures primed withinflammatory-prone hemodynamics derived from the internal carotid sinusand treated with 0.05 ng/ml TNFα deposited a dense fibrin network (1.9e7mean fluorescence intensity) (FIG. 3B; data generated using Protocol Aas described above). Thus, atheroprone hemodynamics prime theendothelial layer to be more responsive to cytokine activation, allowingfor 100- to 200-fold lower levels of TNFα to induce fibrin deposition ascompared to static cultures.

Identical experiments in which only endothelial cells were culturedyielded similar results (data not shown), demonstrating that the resultis hemodynamic-specific and not a consequence of the presence of smoothmuscle cells.

FIG. 4B shows results from experiments performed according to Protocol Cas described above, where shear stress was maintained during clotformation, more closely mimicking physiological conditions. The twoupper panels in FIG. 4B show stacked images for each condition and areslightly angled to show the topography of the clot. The bar graph inFIG. 4B shows the fluorescence intensity of each of these images at theindicated distance above the cell surface. Representative images at 6 μmand 21 μm above the cell surface are shown in the lower right-hand panelof FIG. 4B.

Conclusions:

Static monoculture of endothelial cells requires significantly elevatedlevels of TNFα that are not relevant to human circulating bloodconcentrations in order to induce fibrin deposition. Atheropronehemodynamics up-regulate clotting factors and down-regulate clottinginhibitors compared to atheroprotective hemodynamics. Hemodynamicpriming of endothelial cells in vitro shifts the dose-dependent fibrindeposition response to TNFα into a concentration range similar to thecirculating levels of TNFα observed in humans with cardiovasculardisease, two orders of magnitude below that required to induce fibrindeposition in static endothelial cell cultures.

Example 2: An In Vitro Model for Atherosclerosis

(i) Effects of oxLDL in a Hemodynamic Environment

(a) Methods

To oxidize LDL, native LDL (nLDL) was dialyzed with PBS for 24 hours toremove EDTA. The LDL was then dialyzed for 3 days in PBS containing 13.8μM CuSO₄. The LDL was then dialyzed with PBS containing 50 μM EDTA foran additional 24 hours. A relative electrophoretic migration number wasused to confirm the oxidation level for each batch. Upon completion ofoxidation, the oxLDL was stored under nitrogen at 4° C. until use.

Smooth muscle cells were plated on a first surface of the porousmembrane of a TRANSWELL (polycarbonate, 10 μm thickness and 0.4 μm porediameter, no. 3419, Corning) at a plating density of 20,000 cells/cm²and allowed to adhere to the membrane for two hours. The TRANSWELL wasthen inverted and the cells were incubated in reduced serum growth media(M199 supplemented with 2% FBS, 2 mM L-glutamine, and 100 U/mlpenicillin-streptomycin) for forty-eight hours. Endothelial cells werethen plated on a second surface of the TRANSWELL porous membrane at adensity of 100,000 cells/cm², under the same media conditions andincubated for an additional twenty-four hours prior to the applicationof shear stress.

Following 16-24 hours of shear stress preconditioning withatheroprotective or atheroprone hemodynamic forces, oxLDL was added toupper volume (containing the endothelial cells) at a concentration of10-50 μg/ml (FIG. 5). In devices not receiving oxLDL, nLDL was added tothe upper volume at the same concentration and used as a vehiclecontrol. This concentration of oxLDL is similar to plasma concentrationsof oxLDL observed in patients with cardiovascular disease. Shear stressapplication was continued for an additional 24 hours in the presence ofoxLDL.

Five different donor pairs were used to analyze the oxLDL (50 μg/ml)response compared to nLDL (50 μg/ml) within the atheroprone hemodynamicenvironment in endothelial cells and smooth muscle cells. Uponcompletion of the experiment, RNA was collected for gene array analysis.Significant genes were considered using an FDR of 0.01. Gene expressionresults are reported as the relative expression of the gene as comparedto the expression of β-2 microglobulin (B2M). Protein expression resultsare reported as the relative expression of the protein as compared tothe expression of actin. Activity of NFκB was assessed using anadenovirus NFκB-luciferase (Ad-NFκB-luc) reporter infected in ECs andSMCs.

(b) Results

As shown in FIGS. 6A-6F, the effect of different concentrations of oxLDLon gene expression was compared between the atheroprone hemodynamicenvironment and traditional static cultures. These data were furthercompared to “Healthy” hemodynamic conditions without oxLDL. (In FIGS.6A-6F, “Healthy” indicates the application of atheroprotectivehemodynamics, “Atheroprone” indicates the application of atheropronehemodynamics; and “Traditional” indicates the application of staticculture conditions. mean±SE, n=4, *p<0.05, t-test.) The hemodynamicenvironment significantly regulated many pro- and anti-inflammatorygenes (IL8, E-selectin (SELE), KLF2, eNOS). The response to the additionof oxLDL compared to nLDL created dose-dependent changes in geneexpression that was dependent on the hemodynamic environment.

In particular, previous published studies in traditional static cultureshave shown that HO-1 and ATF3 are “classic” oxLDL-sensitive genes. Asshown in FIGS. 6A and 6B oxLDL activates these genes at much higherlevels under the atheroprone conditions compared to traditional staticconditions.

Unique to atheroprone conditions, oxLDL was also found to activateinflammatory genes such as IL8 and E-Selectin (SELE), which were notregulated in traditional static cultures (FIGS. 6C and 6D).Interestingly, oxLDL reduced atheroprotective signaling (eNOS and KLF2)(FIGS. 6E and 6F).

FIGS. 7A-7E illustrate changes in protein expression in response tooxLDL treatment within the atheroprone hemodynamic environment. Inagreement with gene expression, oxLDL treatment resulted in elevatedVCAM-1 protein expression (pro-inflammatory; FIG. 7A) and reducedphosphorylation of atheroprotective eNOS signaling (FIG. 7B). Further,oxLDL treatment resulted in increased levels of secreted cytokines, suchas IL6, IL8, and MCP-1 (FIGS. 7C-7E). In FIGS. 7A-7E, mean±SE, n=4,*p<0.05, t-test.

Because many of the affected genes and proteins by oxLDL areNFκB-dependent, NFκB activity was assessed using a luciferase reporterin endothelial cells and smooth muscle cells. FIGS. 8A and 8B show thatin endothelial cells (FIG. 8A), atheroprone hemodynamics “prime” thecells for elevated NFκB activity compared to the healthy condition. Thisresponse is further heightened with treatment of oxLDL. Likewise, smoothmuscle cells (FIG. 8B) showed elevated NFκB signaling with treatment ofoxLDL (even though the oxLDL was added to the upper volume, whichcontained only endothelial cells). In FIGS. 8A and 8B, mean±SE, n=4,*p<0.05, t-test.

Full genome arrays were used to interrogate gene expression differencesin ECs and SMCs within the atheroprone condition between 50 μg/ml nLDLand 50 μg/ml oxLDL. FIG. 9 shows heatmaps for gene expression across thetwo different conditions for 5 donors (endothelial cells) or 4 donors(smooth muscle cells). Using stringent statistical cut-off criteria (SAMand wLPE methods), 688 genes were regulated in endothelial cells and 304in smooth muscle cells as compared to cells treated with nLDL. Thesegene panels were enriched with shear stress-regulated, pro- (VCAM,E-Selectin) and anti-(KLF2, eNOS) inflammatory genes.

In sum, oxLDL is known to activate some pathways (HO-1, ATF3), but notothers (E-Selectin, IL6) in traditional static cultures. Here, it wasfound that the addition of oxLDL within the atheroprone hemodynamicenvironment preferentially reduced endothelial “atheroprotective”signaling (KLF2, eNOS expression and phosphorylation), and furtheractivated pro-inflammatory signaling, including adhesion moleculeexpression and cytokine secretion. Though not directly exposed to eitheratheroprone shear stress or LDL, many genes were regulated by theseconditions in the underlying SMCs in this coculture system, includinginflammatory genes. Investigating the role of oxLDL within the contextof physiologic shear stress was found to enhance theatheroprone-regulated gene profile towards a more pro-inflammatoryphenotype. These conditions mimic vessel wall inflammation found inhuman arteries and provide an ideal environment for testing drugsintended for treating advanced atherosclerosis.

(ii) Effects of TNFα in a Hemodynamic Environment

(a) Methods

TNF-α is a potent inflammatory cytokine. The concentration of TNF-α ismodulated by severity of patients with chronic heart failure to levelsof ˜0.02 ng/ml, while in healthy individuals TNF-α is typically muchlower or undetectable. As noted above, circulating levels of TNFαdetected in humans with cardiovascular disease are about 0.01 ng/ml toabout 0.1 ng/ml. In healthy individuals, circulating levels of TNFα aremuch lower or undetectable, for example about 0 ng/ml to about 0.001ng/ml. By comparison, static in vitro experiments typically use TNFαconcentrations of 1-10 ng/ml.

Smooth muscle cells and endothelial cells were plated on first andsecond surfaces of a porous membrane of a TRANSWELL in the same manneras described above for the oxLDL experiments.

Following 24 hours of shear stress preconditioning with atheroprotectiveor atheroprone hemodynamic forces, TNFα was added to upper volume(containing the endothelial cells) at a concentration of 0.05-1 ng/ml(see FIG. 5). This concentration of TNFα is similar to plasmaconcentrations of TNFα observed in patients with cardiovascular disease.Shear stress application was continued for an additional 24 hours in thepresence of TNFα.

(b) Results

As shown in FIG. 10, it was found that hemodynamic priming for 18-24hours sensitized endothelial cells and smooth muscle cells to lowerlevels of TNFα compared to traditional static cultures. Treatingendothelial cells with 0.1-1 ng/ml TNFα induced an inflammatory responsethat was not seen at the same levels in static cultures, as illustratedby increased expression of E-Selectin, ICAM, VCAM, and IL8 in thecultures subjected to atheroprone hemodynamic forces. In FIG. 10,“traditional” indicates that the cells were cultured under staticconditions, and “disease” indicates that the cells were subjected toatheroprone hemodynamic forces. The data in FIG. 10 are shown asmean±SE, n=4, *p<0.05, t-test.

(iii) Effects of oxLDL and TNFα in a Hemodynamic Environment

(a) Methods

To emulate more inflammatory stages of atherosclerosis, it is desirableto combine multiple circulating factors to better emulate the complexityfound within human blood vessels. For these experiments, endothelialcells and smooth muscle cells were plated in the same manner asdescribed above and subjected to atheroprone shear stresspreconditioning for 18-24 hours. The media in the upper volume was thenexchanged for media containing 0.05 ng/ml TNFα and 50 μg/ml of oxLDL.Media containing only 50 μg/ml of nLDL was used as a control.

Following 24 hours of shear stress preconditioning with atheroprotectiveor atheroprone hemodynamic forces, 50 μg/ml of oxLDL and 0.05 ng/ml ofTNFα were added to upper volume (containing the endothelial cells). Thisconcentration of TNFα is similar to plasma concentrations of TNFαobserved in patients with cardiovascular disease. In devices notreceiving oxLDL, nLDL was added to the upper volume at the sameconcentration and used as a vehicle control. Shear stress applicationwas continued for an additional 24 hours in the presence of TNF-α andoxLDL (see FIG. 5). Gene expression analysis was performed using RT-PCR.

(b) Results

The combination of these factors (oxLDL and TNFα) have shown bothsynergistic increases in gene and protein expression, while alsoproviding broader signaling activation than by either oxLDL or TNFαalone. FIG. 11A shows elevated gene expression of E-Selectin (SELE), apro-inflammatory adhesion molecule compared to oxLDL alone. Thus,similar to the results shown in FIGS. 6A-6F, oxLDL causes higher levelsof E-Selectin gene expression than controls treated with nLDL. Theaddition of TNFα with oxLDL caused even greater levels of inflammatorygene expression.

In addition, the atheroprotective signaling through eNOS transcription(NOS3) is strongly reduced in inflammatory conditions. This response wasamplified with the combination of atheroprone shear stress+oxLDL+TNFα(FIG. 11B). Thus, eNOS gene expression (NOS3) was reduced by oxLDL, butwith the combination of TNF-α and oxLDL, the atheroprotective signalingis even further repressed. The end product is a system with a higherbasal level of inflammatory signaling compared to the atheroprone alone.

(iv) Effects of High-Density Lipoprotein (HDL) in a HemodynamicEnvironment

(a) Methods

An additional component of plasma cholesterol distinct from nLDL oroxLDL is HDL. To assess the effects of HDL in the hemodynamicenvironment, endothelial cells and smooth muscle cells were plated asdescribed above and subjected to 24 hours of shear stresspreconditioning with atheroprone hemodynamic forces. HDL was then addedto the upper volume at a concentration of 45-1,000 μg/ml. This broadrange reflects the broad range of HDL concentrations that can existwithin human patients. HDL concentrations in individuals at risk forvascular disease are generally less than about 300 μg/ml, while HDLconcentrations in healthy individuals range from greater than about 300μg/ml up to about 2,000 μg/ml in healthy exercising patients.

In additional experiments, endothelial cell/smooth muscle cellco-cultures plated as described above were preconditioned for 16 hourswith hemodynamic shear stress. From hours 16-24 the cells wereadditionally primed with 0.05 ng/ml TNFα and 50 μg/ml of oxLDL (comparedto a vehicle control containing 50 μg/ml nLDL) in the upper volume. At24 hours, 45 μg/ml or 90 μg/ml HDL was added to the media in the uppervolume and hemodynamic shear stress was continued for the next 24 hours(hours 24-48).

(b) Results

Addition of HDL at 45 μg/ml or 90 μg/ml activates many atheroprotectivegenes while blocking activation of pro-inflammatory genes and proteins.

(v) Effects of Triglycerides Containing Lipoproteins in a HemodynamicEnvironment

Triglycerides (TG) are an important biomarker of cardiovascular disease.Several species of triglyceride-rich lipoproteins (TRLs) including verylow-density lipoprotein (vLDL) and vLDL remnants, as well as chylomicron(CM) remnants appear to promote atherogenesis independently of LDL. TGlevels in healthy patients range from about 40 to about 150 mg/dL. Inpatients with hypertriglyceridemia, TG levels range from greater thanabout 200 mg/dL to about 1500 mg/dL.

Endothelial cell/smooth muscle cell co-cultures can be plated asdescribed above and preconditioned for 24 hours with atheroprone shearstress. TG-containing lipoproteins, containing very low densitylipoprotein (vLDL), chylomicrons (CM), and remnant particles for vLDLand CM can be added to the system at 500 mg/dL, a concentrationrepresentative of levels seen in patients with hypertriglyceridemia.Treatment concentrations of each component are based on the fraction ofTGs each of these components represent: vLDL makes up about 53% of TGs,thus 0.53×500 mg/dL=265 mg/dL; CM makes up about 38% of TGs, thus0.38×500 mg/dL=190 mg/dL for hypertriglyceridemia conditions. This canbe compared to control conditions based on circulating levels oftriglycerides of 150 mg/dL (representative of levels seen in healthypatients) of 80 mg/dL for vLDL and 57 mg/dL of CM. After 24 hours ofhemodynamics preconditioning, vLDL, CM, or vLDL+CM can be added for theremaining 24 hours of the experiment. vLDL and CM remnant-like proteins(RLPs) can be generated by treating the same concentrations listed abovewith Lipoprotein Lipase (LPL). RLPs can be added individually or incombination with vLDL and/or CM.

(vi) Effects of Glucose in Combination with TNFα in a HemodynamicEnvironment

(a) Methods

Diabetes is a disease characterized altered insulin and glucosehomeostasis. In healthy individuals, blood glucose concentrations areabout 5 to about 10 mM, while in diabetic individuals, blood glucoseconcentrations range from greater than about 10 mM to about 20 mM.Diabetes and associated elevated glucose levels are risk factors foratherosclerosis.

Endothelial cells and smooth muscle cells were plated as describedabove. For a period of four days prior to the application of shearstress, the endothelial cells and smooth muscle cells were cultured inthe presence of elevated glucose (15 mM) or basal glucose (5 mM)conditions found in most media formulations, supplemented with mannoseas a vehicle control for glucose to account of potential changes inosmolarity. Cultures were then preconditioned for 24 hours underatheroprone hemodynamics, followed by exposure to 0.05 ng/ml TNFα (aconcentration similar to circulating levels observed in patients withcardiovascular disease) for an additional 24 hours. Upon completion ofthe experiment, RNA was collected for gene expression analysis viaRT-PCR. In some experiments, endothelial cells were infected withadenovirus with NFκB-luciferase construct measuring NFκB activity vialuciferase assay. NFκB activity was assessed as well as pro-inflammatorygenes (E-selectin and ICAM) and anti-inflammatory genes (KLF2 and eNOS).

(b) Results

Cells were chronically exposed to elevated levels of glucose prior toplating for hemodynamic experiments. For the experiment shown in FIGS.12A-12C, endothelial cells were preconditioned using atheropronehemodynamic forces in the presence of elevated glucose for the remainderof the experiment. At the conclusion of the experiment gene expressionand NFκB activity were assessed as a function of the glucose treatmentin combination with TNFα.

While elevated glucose had no effect on basal levels of genes(untreated), samples treated with atheroprone shear stress and TNF-α hadhigher levels of inflammatory signaling when pretreated with elevatedglucose, compared to mannose controls. FIGS. 12A and 12B show thatelevated glucose increased activation of inflammatory signaling,including NFκB (FIG. 12A) activity and downstream gene activation ofadhesion molecules E-Selectin and ICAM (FIG. 12B). Elevated glucose alsocaused larger decreases in atheroprotective signaling (eNOS, KLF2)compared to mannose treated controls (FIG. 12C). The results in FIGS.12A-12C are presented as mean±SE, n=4, *p<0.05, t-test.

Example 3: An In Vitro Model for Hypertension

(i) Angiotensin II (ANG2)

Angiotensin II (ANG2) levels are increased in patients withcardiovascular complications, such as atherosclerosis, diabetes orhypertension. Typical concentrations of ANG2 range from about 1 nM toabout 5 nM in healthy patients, and from greater than about 6 nM toabout 20 nM in hypertensive patients.

To assess the effects of ANG2 in the hemodynamic environment,endothelial cells and smooth muscle cells were plated as described aboveand subjected to 24 hours of shear stress preconditioning with healthyand atheroprone hemodynamic forces. ANG2 at a concentration of 10 nM(10.46 ng/ml) or a DMSO vehicle control (VEH) was added to the uppervolume and RNA was collected for gene array analysis.

Gene array analysis of this condition compared to DMSO vehicle controlsrevealed many significant inflammatory genes that are upregulated byANG2. FIGS. 13A and 13B show gene expression heat maps for bothendothelial cells (FIG. 13A) and smooth muscle cells (FIG. 13B) treatedwith ANG2 under both healthy and atheroprone (disease) conditions. Asseen in FIGS. 13A and 13B, numerous genes were significantly regulatedby ANG2 and the ANG2 conditions sorted together in an unbiased way.

(ii) Aldosterone

Aldosterone is an important signaling hormone downstream of ANG2 in therenin-angiotensin system. Its levels can vary under a number ofpathologies, including atherosclerosis, diabetes, and hypertension.Concentrations of aldosterone in healthy individuals are about 0.3 mM.Concentrations of aldosterone in individuals with hperaldosteronismrange from about 0.8 mM to about 1 mM.

To assess the effects of aldosterone in the hemodynamic environment,endothelial cells and smooth muscle cells were plated as described aboveand subjected to 24 hours of shear stress preconditioning with healthyand atheroprone hemodynamic forces. Aldosterone at a concentration of 1mM or a vehicle control (VEH) was added to the upper volume and RNA wascollected for gene array analysis.

Gene array analyses of this condition compared to DMSO vehicle controlsreveal many significant inflammatory genes that are upregulated byaldosterone. FIGS. 14A and 14B show the gene expression heat maps forendothelial cells and smooth muscle cells treated with aldosterone(Aldo) in both healthy and atheroprone (disease) conditions. Manysignificant genes were regulated by these conditions, with the majorityof regulated genes found within the atheroprone hemodynamic environment.

Example 4: A Physiologic In Vitro Liver Model

Static hepatocyte cell culturing methods are associated with poor invitro to in vivo correlations, due in part to the absence ofphysiological parameters which maintain metabolic phenotype over time invivo. The inventors have now discovered that restoring physiologicalhemodynamics and transport retains hepatocyte phenotype and function invitro compared to the standard static hepatocyte collagen gelconfiguration.

To recreate a cellular hepatocyte system with fluid dynamics andtransport analogous to in vivo liver circulation, a cone-and-platedevice-based technology was employed that has been extensively used tore-establish in vivo blood vessel cell phenotypes by recreating theexposure of vascular endothelial cells to human-derived hemodynamicblood flow forces in vitro. This technology is described in U.S. Pat.No. 7,811,782, the contents of which are hereby incorporated byreference. The technology (FIG. 15B) was adapted and modified to designa rat liver monoculture system which applies hemodynamic flow andtransport conditions reflective of in vivo hepatic circulatory values.The configuration of cells in the device (FIG. 15C) is based on in vivomicroarchitecture of hepatic lobules (see FIG. 15A) where cords ofhepatocytes are separated from sinusoidal blood flow by a filteringlayer of endothelial cells. This design uses a porous polycarbonatemembrane suspended in a cell culture container, with primary rathepatocytes sandwiched in a collagen gel on one side of the porousmembrane. The porous membrane acts analogously to the filtering layer ofsinusoidal endothelial cells which is present in the liver. Media iscontinuously perfused on both sides of the porous membrane, whilehemodynamic forces, derived from a range of physiological blood flowvalues, are continuously applied to the non-cellular side of the porousmembrane. The entire set up is housed in a controlled environment with5% CO₂ and at 37° C. A flow-based culture system was effectively createdwhereby hepatocytes are shielded from direct effects of flow, as theywould be in vivo. Recapitulating the hemodynamics and in a systemdesigned to be analogous to the microstructure of the hepatic sinusoidresults in stable retention of a differentiated hepatic and metabolicphenotype similar to that of in vivo liver.

Methods

(i) Animal Surgery and Hepatocyte Isolation

All animals used for the experiments were treated according to protocolsapproved by HemoShear's Animal Care & Use Committee. Hepatocytes wereisolated from male Fischer rats (250 g-350 g) by a modification ofSeglen's two-step collagenase perfusion procedure using a 20 mL/min flowrate (Seglen, Hepatocyte Suspensions and Cultures as Tools inExperimental Carcinogegnesis, J. Toxicology & Environmental Health,5(2-3): 551-560 (1979), the contents of which are hereby incorporated byreference). Briefly, the rats were anaesthetized with isoflurane,following which the abdominal cavity was incised and the inferior venacava was canulated while making an excision was made in the portal veinfor outflow. The liver was perfused in two steps, first with a Ca⁺⁺-freebuffer to flush out blood and break up intercellular junctions, followedby collagenase in a Ca⁺⁺-containing buffer to digest the extracellularcollagen matrix. After the liver was suitably perfused it was excisedand freed of the capsule in a Petri dish under a sterile hood. Anenriched hepatocyte population (˜95% purity) was obtained by twosequential 65 g centrifugation and washing cycles of 10 minutes eachfollowed by a 10 minute spin with 90% PERCOLL (colloidal silicaparticles of 15-30 nm diameter (23% w/w in water) coated withpolyvinylpyrrolidone (PVP); used to establish density gradients that canbe used to isolate cells). The viability of hepatocytes was determinedby trypan blue exclusion test and cells with a viability over 85% areused.

(ii) Cell Culture and Device Operating Conditions

Hepatocyte Culture Media: For the data shown in FIGS. 16A-16F, 17A-17C,18A, 18B, 19A-19D, and 20A-20C, the rat hepatocyte culture mediacontained base media of DMEM/F12 containing high glucose (17.5 mM),supplemented by fetal bovine serum (10% at the time of plating andreduced to 2% for maintenance after 24 hours). The media also containedgentamycin (50 μg/ml), ITS (insulin concentration 2 μMol), 1% NEAA, 1%GLUTAMAX, and dexamethasone (1 μM at plating and 250 nM for maintenanceafter 24 hours).

Collagen coating and plating: Collagen solution was made by mixing TypeI Rat Tail Collagen in sterile distilled water, 10× phosphate bufferedsaline (PBS) and 0.2N sodium hydroxide in a predefined ratio (To make up1 ml, the components were 440 μl, 375 μl, 100 μl and 85 μlrespectively).

For cultures to be subjected to static conditions, 100 mm tissueculture-treated sterile cell culture dishes were coated with 7 μl/cm² ofcollagen solution. For cultures to be subjected to controlledhemodynamics, the lower surface of the porous membrane of 75 mmTRANSWELLS (polycarbonate, 10 μm thickness and 0.4 μm pore diameter, no.3419, Corning) were coated with 7 μl/cm² of collagen solution. Afterallowing an hour for the solution to gel, the surfaces were washed withDPBS, hepatocytes were plated at a seeding density of 125,000 viablecells/cm², and a second layer of collagen gel added after 4 hours. After1 hour, the TRANSWELLS were inverted and placed into cell culturedishes, and media was added (9 ml in the lower volume and 6 ml in theupper volume). 7 ml of media was added to the tissue culture dishes tobe used for static cultures. After 24 hours, the media was switched tomaintenance media (containing 2% FBS), and the cell culture dishescontaining TRANSWELLS were placed into the cone-and-plate device.Controlled hemodynamics were applied to the surface of the porousmembrane of the TRANSWELL in the upper volume.

Operating conditions: The shear stress in dynes/cm² (τ) was calculatedfor a typical hepatic sinusoid based on the formula for pressure drivenflow of a Newtonian fluid through a cylinder,

$\tau = \frac{\Delta \; {P \cdot r}}{2l}$

using reference values for pressure gradient across the sinusoid (ΔP),radius of sinusoids (r) and length of the sinusoids (l) from theliterature. As part of an initial optimization process, a range ofapplied shear stress conditions obtained by altering media viscosity andcone speed that resulted in rates within an order of magnitude of thevalue predicted from literature were seen to be correlated withdifferent transport profiles of horse radish peroxidase dye across themembrane. These were tested for gene expression profiles of thehepatocytes 7 days into culture (data not shown). No differences wereobserved between static cultures and those that were simply perfusedwithout any applied shear and based on the gene expression profiles, anoperational shear rate of 0.6 dynes/cm² was selected for all theexperiments described in this Example.

(iii) Assessment of Phenotypic, Functional, and Metabolic Parameters

RT-PCR: Changes in metabolic and insulin/glucose/lipid pathway geneswere assessed by extracting RNA from hepatocytes from devices run underhealthy and steatotic conditions at the end of the culture period (7 or14 days) and performing RT-PCR on this RNA. The TRANSWELLS were areremoved from the devices and washed with PBS prior to scraping the cellsoff the porous membrane. Total RNA was isolated using a PURELINK RNAMini Kit (a kit for purification of total RNA from cells) and reversetranscribed to cDNA using the ISCRIPT cDNA Synthesis Kit (a cDNAsynthesis kit). Primers were designed for the metabolic genes CYP1A1,CYP1A2, CYP3A2, MDR, and GST as well as the insulin/glucose/lipidpathway genes GPAT, ACC1, IRS-2, PPAR-γ, SREBP, ChREBP, LXR, SCD1, CPT1.Primer sequences are shown below in Table 1:

TABLE 1 Rat Primer Sequences Gene Forward (SEQ ID NO.)Reverse (SEQ ID NO.) CYP1A1 GCTGCTCTTGGCCGTCACCA (1)TGAAGGGCAAGCCCCAGGGT (2) CYP1A2 CCTGCGCTACCTGCCCAACC (3)GGGCGCCTGTGATGTCCTGG (4) CYP3A2 CGGCGGGATTTTGGCCCAGT (5)CAGGCTTGCCTGTCTCCGCC (6) MDR GCTGCTGGGAACTCTGGCGG (7)CCGGCACCAATGCCCGTGTA (8) GST (Pi CGCAGCAGCTATGCCACCGT (9)CTTCCAGCTCTGGCCCTGGTC (10) subunit) GPAT AGCGTTGCTCCATGGGCATATAGT (11)TGTCAGGGATGGTGTTGGATGACA (12) ACC1 TGTCATGGTTACACCCGAAGACCT (13)TTGTTGTTGTTTGCTCCTCCAGGC (14) IRS-2 GCGAGCTCTATGGGTATATG (15)AGTCCTCTTCCTCAGTCCTC (16) PPAR-g ATATCTCCCTTTTTGTGGCTGCTA (17)TCCGACTCCGTCTTCTYGATGA (18) SREBP GGAGCCATGGATTGCACATT (19)AGGCCAGGGAAGTCACTGTCT (20) ChREBP CTATGTCCGGACCCGCACGC (21)CTATGTCCGGACCCGCACGC (22) LXR ACTCTGCAACGGAGTTGTGGAAGA (23)TCGGATGACTCCAACCCTATCCTT (24) SCD1 TGTGGAGCCACAGGACTTACAA (25)AGCCAACCCACGTGAGAGAAGAAA (26) CPT1 ATGTGGACCTGCATTCCTTCCCAT (27)TTGCCCATGTCCTTGTAATGTGCG (28) CYP2B1 GAGGAGTGTGGAAGAACGGATTC (29)AGGAACTGGCGGTCTGTGTAG (30) CYP2B2 TCATCGACACTTACCTTCTGC (31)AGTGTATGGCATTTTGGTACGA (32)RNA expression was analyzed by real-time RT-PCR using IQ SYBR GreenSupermix (a PCR reagent mixture for RT-PCR) and a CFX96 Real-Time Systemwith C1000 Thermal Cycler (an RT-PCR detection system and thermalcycler). RNA data were normalized to endogenous expression ofβ2-microglobulin and reported as a relative quantity compared to healthycultures.

Urea and Albumin Assays: Media collected from static cultures anddevices at various time points was assayed for albumin using arat-specific ELISA based kit (Bethyl Laboratories) as per themanufacturer's protocols. Urea was estimated from the media samplesusing a standard colorimetric assay (QUANTICHROM Urea Assay Kit,DIUR-500, Gentaur). All measurements between the systems were normalizedto a per million cells/day rate for comparison based on the volume ofmedia perfused and the number of initially plated cells.

Western Blots: Following application of controlled hemodynamics, ⅓ ofthe plated surface of the porous membrane of the TRANSWELL (˜1.8 millioncells) was harvested for protein in 150 μl 1×RIPA buffer containingfresh 150 mM DTT and protease inhibitors (HALT Protease InhibitorCocktail (Pierce)+1 mM PMSF+200 mM DTT). Samples were sonicated on icewith 5×1 second pulses, allowed to sit on ice for 30 minutes andcentrifuged at 17,000×g for 10 minutes in a chilled microcentrifuge.Protein determination was done using A660 nm Protein Reagent (Pierce).Samples were boiled 70° C. for 10 minutes and then run on a 7.5% TGX gel(a pre-cast polyacrylamide gel, BioRad) before wet-transferring to 0.2μm PVDF membrane and blocking in 5% non-fat milk at room temperature for10 minutes. Membranes were incubated overnight at 4° C. in rabbit antiUGT antibody (Cell Signaling, 1:500 dilution). Secondary antibody (SantaCruz, Goat anti Rabbit HRP, 1:5000 dilution) incubation was at roomtemperature for one hour. Chemiluminescent signal was developed usingSUPERSIGNAL WEST PICO (a chemiluminescent substrate for horseradishperoxidase, Pierce) reagent and captured using an Innotech ALPHAEASEimaging system. For normalization, gels were probed for mouse antiβ-Actin (Sigma A1978, 1:2000 dilution) followed by secondary goat antimouse HRP (Santa Cruz sc-2005, 1:10,000 dilution).

Immunostaining and Biliary Activity Stain: Antibodies used: Hnf4a (SantaCruz sc-8987), E-cadherin (Santa Cruz sc-71009), and anti-MRP2 (Abcamab3373). At the chosen time points in the experimental design, thestatic cultures and cultures subjected to controlled hemodynamics werewashed gently with 1×PBS, following which they were fixed with 4%paraformaldehyde for 30 minutes. The samples were stored in PBS at 4° C.until they were to be immunostained. For immunostaining, the sampleswere first permeabilized with 0.1% TRITON X (a nonionic surfactant) for20 minutes and then washed with PBS and blocked with 5% goat serum. Theincubation with primary antibodies was at a dilution of 1:100 for 1hour. After 3 washes with PBS with 1% BSA, the secondary antibody wasadded at a dilution of 1:500 for another hour. The samples were thenwashed with PBS plus 1% BSA and then mounted for confocal imaging.

For imaging of the biliary activity at canalicular junctions, sectionsof the porous membrane of the TRANSWELL were washed with PBS andincubated with media containing 10 μM carboxy-2,7-dichlorofluoresceindiacetate (CDFDA) for 10 minutes. Samples were then washed with PBS andplaced on glass slide for confocal imaging.

Transmission Electron Microscopy: Transmission electron microscopy wasperformed as described below in Example 5.

Cytochrome Activity Assays: Hepatocytes were cultured in thecone-and-plate devices under static or controlled hemodynamic conditionsfor five days, and then treated with 0.1% dimethyl sulfoxide (DMSO) orknown inducers of cytocrhome enzymes(3-methylcholanthrene anddexamethasone) for 48 hours. Porous membrane segments roughly 2 cm² inarea were excised and transferred to standard 24-well plates alongsidecorresponding static cultures. The cells were incubated with 500 μl ofhepatocyte media containing substrates from commercially availableP450-GLO kits (kits for luminescent cytochrome p450 assays) at themanufacturer-recommended concentrations. After 4 hours, the media wastransferred to 96-well plates and assayed for luminescent metabolites toreflect cytochrome p450 activity as per the manufacturer protocol. TheATP content of the cells in the same porous membrane segments or staticwells was then estimated by the CELLTITER-GLO assay (a kit for aluminescent cell viability assay) using the manufacturer's protocol, andthe cytochrome values were normalized to ATP content.

Results (i) Controlled Hemodynamics Maintain Hepatocyte Phenotype,Polarized Morphology and Transporter Localization Relative toTraditional Static Monoculture Conditions.

Freshly isolated rat primary hepatocytes were obtained and plated incollagen gel sandwiches on porous membranes. After 1 day, cultures wereeither continued under standard static conditions in a CO₂ incubator at37° C. or introduced into the hemodynamic flow technology and maintainedunder controlled hemodynamics at pre-determined indirect shear rates of0.6 dynes/cm². Media was changed every 48 hours in static cultures andthe devices were continuously perfused. After 7 days, the cultures wereremoved and fixed with 4% paraformaldehyde before immunostaining withantibodies for the hepatocyte differentiation markers E-cadherin andHNF-4α, and visualized by confocal microscopy. E-cadherin stainingpatterns in static collagen gel sandwich cultures (FIG. 16A) displayedhigher levels of cytoplasmic E-cadherin confirmed and quantified bymorphometric analysis (adjacent graphs) and disrupted peripheralmembrane distribution. Under controlled hemodynamics (FIG. 16B),hepatocytes exhibited a more differentiated morphology characterized bydistinct peripheral membrane localization and lower cytoplasmic levelsof E-cadherin. The staining pattern of the HNF4α showed a distinctdifference in localization patterns with the cells in static cultureshaving a more diffuse staining pattern by 7 days (FIG. 16C) while thecells under controlled hemodynamics retained staining confined to thenucleus (FIG. 16D), similar to what is seen in vivo. Polarizedmorphology and canalicular localization of the transporter multi drugresistant protein-2 (MRP-2) that appears after 5-7 days of culture incollagen gel sandwiches is lost in static cultures by day 14 (FIG. 16E)but the canalicular network patterns are stable and extensive undercontrolled hemodynamics (FIG. 16F). Day 14 cultures maintained undercontrolled hemodynamics co-stained for MRP-2 and HNF-4α (FIG. 17A)alongside sections from rat in vivo liver (FIG. 17B) show very similarstaining patterns. Transmission electron microscopy images of day 7cultures under controlled hemodynamics (FIG. 17C) demonstrate theretention of subcellular components such as rough and smooth endoplasmicreticulum and mitochondria in addition to confirming the presence ofbile canaliculi and tight junctions.

(ii) Controlled Hemodynamics Results in Retention of Hepatocyte-SpecificFunction in Rat Hepatocytes in a Collagen Gel Configuration Relative toStatic Cultures Over 14 Days.

Hepatocytes were cultured under static or controlled hemodynamics (0.6dynes/cm²) for 2 weeks and media sampled at 4, 7, 11, and 14 days.Assays for urea and albumin were performed on the media and the valueswere normalized to production rates over 24 hours per million cellsbased on the initial number of plated cells. Hepatocyte functionreflected by secreted albumin estimated from media samples at varioustime points over 14 days and expressed as μg/10⁶ plated hepatocytes/day(FIG. 18A), showed significantly higher levels (3-4 fold) undercontrolled hemodynamics (solid line) as compared to static cultures(dashed line) (Day 7: 97.96±11.34 vs. 25.84±8.22, p=0.00001; Day 14:87.80±8.62 vs. 33.93±4.39, p=0.0001). Urea secretion (FIG. 18B) byhepatocytes expressed as μg/10⁶ plated hepatocytes/day under controlledhemodynamics (solid line) was also found to be at 4-5 fold higher levelsthan static cultures (dashed line) consistently over two weeks inculture (Day 7: 622.78±33.96 vs. 139.76±13.37, p=2.7×10⁻⁹; Day 14:667.71±84.37 vs. 178.68±6.13, p=1×10⁻⁶).

(iii) Controlled Hemodynamics Differentially Regulates the Expression ofPhase I and Phase II Metabolic Genes and Proteins Compared to StaticCultures.

Hepatocytes were cultured under static or controlled hemodynamics (0.6dynes/cm²) for 7 days. QRT-PCR was performed for select metabolic genes(Table 1) on RNA samples at day 7 from these conditions. All values werenormalized to day 7 static cultures. Hepatocytes cultured undercontrolled hemodynamics resulted in gene expression levels that wereconsistently higher than in static cultures (n=11, Fold changes relativeto static cultures: Cyp1A1 ˜54, p=0.0003; Cyp1A2 ˜64, p=0.005, Cyp2B1˜15, p=0.001: FIG. 19A, Cyp2B2 ˜2.7, p=0.09 and Cyp3A2 ˜4, p=0.075: FIG.19B) and closer to in vivo levels. Interestingly, the expression levelsof the gene for the Pi subunit of phase II enzyme GST, known to increasein static cultures over time, was lower in both in vivo liver (−4.9fold, p=0.152) and hepatocytes cultured under controlled hemodynamics(−2.3 fold, p=0.025) compared to static cultures (FIG. 19C).

Hepatocytes were cultured under static or controlled hemodynamics (0.6dynes/cm²). Cell cultures were taken down at 4, 7, 11 and 14 days andcell lysates were obtained as described in the methods section,normalized to total protein, and equivalent samples were loaded and runon SDS page gels before probing with antibodies for the phase II enzymeUGT1 A1 and β-actin (for normalization). Western blots (FIG. 19D)demonstrate that UGT1 A1 is upregulated under controlled hemodynamics ascompared to static conditions at all the time points over 2 weeks inculture. In the same experiment, part of the porous membrane of theTRANSWELL from 14 day cultures under controlled ahemodynamics was fixedwith 4% paraformaldehyde and stained for HNF-4a and the canaliculartransporter protein MRP-2, demonstrating retention and localization ofMRP-2 along the canalicular junctions between the hepatocytes (FIG.17A). The remainder of the membrane was excised after removal from thedevice and immediately incubated with the substratecarboxy-2,7-dichlorofluorescein diacetate (CDFDA). The cells were imagedby confocal microscopy over a time window of 20 minutes to observe thebreakdown of the substrate into carboxy-2,7-dichlorofluorescein (CDF)and its active secretion into the bile canalicular structures (seen inFIG. 17C). The pattern was very similar to that of sectioned samples ofin vivo liver immunostained with antibodies to MRP-2 and HNF-4a (FIG.17B).

(iv) Rat Hepatocytes Cultured Under Controlled Hemodynamics Display aHigher Level of Basal and Inducible Cytochrome p450 Activity than StaticCultures at More In Vivo-Like Concentrations

To validate that the increase in metabolic genes and proteins translatedto changes in metabolic activity, primary rat hepatocytes were culturedas described earlier in the cone-and-plate devices under controlledhemodynamics (0.6 dynes/cm²) and in static collagen gel cultures. After5 days, they were either left untreated or treated with 0.1% DMSO, 1A/1Binducer 3-Methyl Cholanthrene (3-MC, 1 μM in static and 0.1 μM undercontrolled hemodynamics) or 3A inducer dexamethasone (50 μM in staticand 02.5 μM under controlled hemodynamics). After 48 hours, on day 7,segments of the porous membrane from the devices containing hepatocytescultured under controlled hemodynamics that were roughly 2.0 cm² in areawere excised and transferred to standard 24-well plates and treated withsubstrates for the Cyp p450 enzymes in parallel to corresponding staticcultures treated with the different agents. Cytochrome p450 assays weredone on day 7 using commercially available P450-GLO kits. After 4 hoursthe media was transferred to 96-well plates and assayed for luminescentmetabolites to reflect cytochrome p450 activity. Values were normalizedto the ATP content of the cells assessed by CELLTITER-GLO assay in orderto get an accurate representation of live cells and avoid anyconfounding effects of the collagen gels on total protein measurements.

Basal activity level of the cytochrome p450 enzymes (FIG. 20A) inuntreated cultures was upregulated by controlled hemodynamics comparedto static (1A˜15 fold, 1B˜9 fold and 3A˜5 fold). In spite of higherlevels of basal activity, under controlled hemodynamics the response toclassical inducers (FIG. 20B) was well maintained (1A/1B response toDMSO vs. 3-MC—4.87 vs. 133.06; 3A response to DMSO vs.Dexamethasone—11.64 vs. 57.53).

While initially measuring the Cyp activity to confirm the enhanced geneexpression that was noted under controlled flow, 50 μM dexamethasone,the concentration recommended for inducing static cultures, was toxic inthis system. As a result the concentration of the dexamethasone wasdecreased to 1 μg/ml in order to get an inductive response, a level thatcorrelates well with plasma concentrations seen in vivo in rats.Similarly, induction responses for 3-MC were also seen at 10-fold lowerlevels under controlled hemodynamics

To confirm the presence of transporter activity under controlledhemodynamics, TRANSWELL filter segments from the devices were incubatedwith the substrate carboxy-2,7-dichlorofluorescein diacetate (CDFDA).The compound was broken down to the fluorescent form CDFCarboxy-2,7-Dichlorofluorescein which was actively secreted out into thecanalicular spaces demonstrating active canalicular transport (FIG.20C).

The data described above are the result of experiments carried out toevaluate the effect of exposing hepatocytes to controlled hemodynamicsin order to restore their phenotype more similar to that observed invivo. These experiments used standard media formulations routinely usedin static culture in order to allow for side by side comparison with thestatic collagen gel cultures and identify the selective benefits ofcontrolled hemodynamics. In the course of these experiments, hepatocytescultured under these controlled hemodynamic conditions demonstratedenhanced in vivo-like phenotype and function and were more responsive toinducers such as dexamethasone and 3-MC. However, some accumulation oflipids was also observed in hepatocytes cultured with the concentrationsof glucose (17.5 mM) and insulin (2 μMol) which are used routinely forassays in static systems. It has now been discovered that whenhepatocytes are cultured under controlled hemodynamic conditions asdescribed herein, much lower concentrations of glucose and insulin,similar to the concentrations observed in healthy individuals in vivo,can be used. The data indicate that these lower concentrations ofglucose (5.5 mM) and insulin (2 nM) further enhance hepatocyte functionand metabolic activity. Moreover, hepatocytes can be cultured undercontrolled hemodynamics in media containing the higher concentrations ofglucose and insulin in order to create a model of fatty liver disease,as explained further in the following Example.

Example 5: An In Vitro Model for Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is the most common cause ofliver dysfunction and is associated with obesity, insulin resistance,and type 2 diabetes. The changes in the fatty liver progress from earlyaccumulation of fat vesicles within hepatocytes (hepatic steatosis) tosubsequent loss of liver metabolic function and inflammatory changes,ultimately leading to fibrosis and cirrhosis. Animal in vivo models offatty liver disease have successfully used either high fat diets or lowfat, high carbohydrate diets that induce the hyperglycemia andhyperinsulinemia reflective of the diabetic milieu to inducetriglyceride buildup. However in vitro models typically use onlyoverloading with free fatty acids (oleic, palmitic or linoleic acid) toinduce fatty changes and may not capture the de novo hepatocyte responseto the high levels of glucose and insulin that may play a critical rolein the pathogenesis of the disease. Static hepatocyte cultures are alsoknown to have a markedly decreased insulin response and standard culturemedias typically require high non-physiological levels of the hormonefor basic hepatocyte survival and function. The model described herein,by contrast, preserves a more physiological hepatocyte response to drugsand hormones and allows us to maintain basic liver function at closer toin vivo concentration levels of glucose and insulin (as described abovein Example 4), and furthermore allows us to elicit the pathologicresponse seen in fatty liver by creating a diabetic-like milieucharacterized by high glucose and insulin levels.

Methods:

(i) Animal Surgery and Hepatocyte Isolation

Animal surgery and hepatocyte isolation were performed as describedabove in Example 4.

(ii) Cell Culture and Device Operating Conditions

Healthy hepatocyte culture media: The healthy hepatocyte culture mediacontained base media of DMEM/F12 containing low glucose (5.5 mM),supplemented by fetal bovine serum (10% at the time of plating andreduced to 2% for maintenance after 24 hours). Additionally, the mediacontained gentamycin (50 μg/ml), ITS (insulin, transferrin, andselenium; insulin concentration of 2 nM), 1% non-essential amino acids(NEAA), 1% GLUTAMAX (a media supplement containingL-alanyl-L-glutamine), and dexamethasone (1 μM at plating and 250 nM formaintenance after 24 hours for the data shown in FIGS. 21 and 22; 100 nMthroughout the experiment for the data shown in FIGS. 25A, 25B, 26, 27A,27B, 28A, 28B, 29A, 29B, 30A-30C, 31A, and 32).

Media to induce fatty liver changes (“fatty liver media”): The culturemedia used to induce fatty liver changes contained base media ofDMEM/F12 containing high glucose (17.5 mM), supplemented by fetal bovineserum (10% at the time of plating and reduced to 2% for maintenanceafter 24 hours). The media also contained gentamycin (50 μg/ml), ITS(insulin concentration 2 μMol), 1% NEAA, 1% GLUTAMAX, and dexamethasone(1 μM at plating and 250 nM for maintenance after 24 hours for the datashown in FIGS. 21 and 22; 100 nM throughout the experiment for the datashown in FIGS. 25A, 25B, 26, 27A, 27B, 28A, 28B, 29A, 29B, 30A-30C, 31,and 32).

Collagen coating and plating: Collagen solution was made as describedabove in Example 4. The lower surfaces of the porous membranes of 75 mmTRANSWELLS (polycarbonate, 10 μm thickness and 0.4 μm pore diameter, no.3419, Corning) were coated with 300 μl of the collagen solution. Afterallowing an hour for the solution to gel, the surfaces were washed withDPBS, hepatocytes were plated at a seeding density of 125,000 viablecells/cm², and a second layer of collagen gel added after 4 hours. After1 hour, the TRANSWELLS were inverted and placed into cell culturedishes, and media was added (9 ml in the lower volume and 6 ml in theupper volume). After 24 hours (i.e., on day 2 of the experiments), themedia was changed to maintenance media (the healthy or fatty liver mediadescribed above) and the Petri dishes were placed in the cone-and-platehemodynamic flow device, and controlled hemodynamics were applied to thesurface of the porous membrane of the TRANSWELL in the upper volume. Insome experiments, the maintenance media contained 1.5 μM pioglitazone in0.1% DMSO vehicle or the 0.1% DMSO vehicle alone. The cells werecultured under controlled hemodynamics until day 7, when hepatocyteswere examined using the assays described below.

Operating conditions: The shear stress was calculated as described abovein Example 4. A range of applied shear stress conditions, generated byaltering media viscosity and cone speed, and resulting in rates withinan order of magnitude of the value predicted from literature (0.1 to 6dynes/cm²) were used. These were correlated with different transportprofiles of reference dye horse radish peroxidase dye across themembrane. Cultures were run for 7 days and assessed for fatty liverchanges.

(iii) Measurement of Fatty Liver Changes:

To examine changes occurring in the fatty liver model against healthycontrols the following were evaluated:

(a) Changes in metabolic and insulin/glucose/lipid pathway genes(RT-PCR);(b) Accumulation of intracellular lipids within hepatocytes by Oil Red Oassay, Nile red staining, and measurement of total triglycerides;(c) Changes in differentiated function of hepatocytes (urea and albuminsecretion);(d) Changes in metabolic activity (Cytochrome p450 assays); and(e) Morphological changes within hepatocytes by transmission electronmicroscopy (TEM).

RT-PCR and urea and albumin assays were performed as described above inExample 4.

Staining Methods: Hepatocyte TRANSWELL membrane sections werepermeabilized in 0.1% Triton-X diluted in PBS for 20 minutes and washedthrice in PBS for five minutes each. Samples were then blocked in 5%goat serum, 0.2% blotting grade non-fat dry milk blocker, and 1% BSA inPBS for 45 minutes. The samples were then washed thrice in 0.1% BSA inPBS and incubated with 1:5000 dilution of Nile red (1 mM stock), 1:1000DRAQ5 (a fluorescent DNA dye; Cell Signalling), 1:500 ALEXA FLUOR 488conjugated phalloidin (Life Technologies), and 1% BSA in PBS for thirtyminutes and protected from light. The samples were washed in 0.1% BSA inPBS thrice for five minutes each and mounted on glass cover slips usingPROLONG GOLD antifade mounting media (an antifade reagent; Invitrogen).The samples were imaged on a Nikon C1+Confocal System microscope.

Transmission Imaging Microscopy (TEM): Segments of the porous membranesfrom TRANSWELLS containing hepatocytes cultured under healthy orsteatotic conditions for 7 days were washed with PBS before fixing in asolution containing 4% paraformaldehyde and 2% glutaraldehyde for 1hour. The samples were then sent to be processed for TEM at theUniversity of Virginia imaging center. TEM images were evaluated foraccumulation of lipid within the hepatocytes, the appearance ofsubcellular organelles such as mitochondria and smooth and roughendoplasmic reticulum, retention of polarized morphology, and bilecanaliculi.

Oil Red O Assay: Accumulation of intracellular lipids within hepatocyteswas assessed by adapting and modifying a commercially availableSteatosis Colorimetric Assay Kit (Cayman Chemical). At the end of theculture period, 2 cm² sized porous membrane segments containing thehepatocytes from devices under healthy and steatotic conditions werewashed with PBS and fixed in 4% paraformaldehyde for 30 minutes. Theseporous membrane segments were then washed with PBS, dried completely andincubated with 300 μl of Oil Red O working solution for 20 minutes in 24well plates. The porous membrane segments were then washed repeatedlywith distilled water 7-8 times followed by two five minute washes withthe wash solution provided in the Steatosis Colorometric Assay Kit. Dyeextraction solution (300 μl) was added to each well and the plates wereincubated on an orbital shaker for 15-30 minutes under constantagitation. The solution was then transferred to clear 96-well plates andabsorbance was read at 490-520 nm in a spectrophotometer.

Measurement of Total Triglycerides: Triglyceride content was assessedusing a commercially available colorimetric assay kit (CaymanTriglyceride Colorimetric Assay Kit, Cat #10010303). At the end of thetreatment period, cells were collected from the porous membranes byscraping with a rubber policeman and PBS, after which they werecentrifuged (2,000×g for 10 minutes at 4° C.). The cell pellets wereresuspended in 100 μl of cold diluted Standard Diluent from thetriglyceride assay kit and sonicated 20 times at one second bursts. Thecell suspension was then centrifuged at 10,000×g for 10 minutes at 4° C.The supernatant was removed and used for the assay as per themanufacturer's protocol and normalized to protein content from the samesamples.

Cytochrome Activity Assays: Hepatocytes were cultured in thecone-and-plate devices under healthy and steatotic conditions for 7days. Porous membrane segments roughly 2 cm² in area were excised andtransferred to standard 24-well plates alongside corresponding staticcultures. The cells were incubated with 500 μl of healthy hepatocytemedia containing substrates from commercially available P450-GLO kits atthe manufacturer-recommended concentrations. After 4 hours, the mediawas transferred to 96-well plates and assayed for luminescentmetabolites to reflect cytochrome p450 activity as per the manufacturerprotocol. The ATP content of the cells in the same porous membranesegments or static wells was then estimated by the CELLTITER-GLO assayusing the manufacturer's protocol, and the cytochrome values werenormalized to ATP content.

Results:

Nile red staining: FIGS. 25A and B show staining of hepatocytes culturedin the healthy (FIG. 25A) or fatty liver (FIG. 25B) media with Nile red,phalloidn, and DRAQ5. As can be seen in FIG. 25B, the hepatocytescultured in the fatty liver media (containing high concentrations ofglucose and insulin) accumulate a large number of lipid droplets.

Transmission electron microscopy: Hepatocytes cultured in the fattyliver media were also examined by transmission electron microscopy. Asshown in FIG. 26, hepatocytes cultured under these conditions accumulatelipid. A large lipid droplet is indicated in the hepatocyte on the leftside of the image. Gap junctions between two hepatocytes are also shown,demonstrating the polarized morphology.

Total lipid and total triglycerides: As shown in FIGS. 27A and 27B,total lipid (FIG. 27A) and total triglycerides (FIG. 27B) were bothsignificantly increased in hepatocytes cultured under the highglucose/high insulin fatty liver conditions in the presence ofliver-derived hemodynamics. Oil red O quantification indicated that thetotal lipid was raised in the disease cultures by about 3-fold ascompared to the healthy cultures.

Gene expression: Glycerol 3-phosphate acyltransferase (GPAT) is a keyenzyme involved in triglyceride synthesis and known to upregulated andcontribute to steatosis and fatty liver. As shown in FIG. 21, primaryrat hepatocytes cultured under controlled hemodynamics in the deviceswhen exposed to pathological conditions (n=9) of high insulin (2 μMol)and high glucose (17.5 mMol) exhibit a significantly higher expressionthe GPAT gene (p=0.04) compared to those cultured under healthyphysiological levels (n=6) of insulin (2 nMol) and glucose (5.5 mMol) inthe media. The results are expressed as fold increase over standardstatic cultures in collagen gel sandwiches (2 μMol insulin and 17.5 mMolglucose).

Similar results are shown in FIG. 28B for hepatocytes cultured undercontrolled hemodynamics in healthy or fatty liver media containing alower concentration of dexamethasone. The hepatocytes cultured in thehigh insulin/high glucose (fatty liver) media exhibited significantlyhigher levels of GPAT expression as compared to hepatocytes cultured inthe healthy media containing lower levels of insulin and glucose. Asshown in FIG. 28A, hepatocytes cultured under controlled hemodynamics inthe high insulin/high glucose media also exhibited significantly higherlevels of expression of sterol regulatory element-binding protein(SREBP), another key gene responsible for lipogenisis, as compared tohepatocytes cultured in the healthy media.

These steatotic changes were accompanied by concomitant metabolicchanges. Of all the key metabolic enzymes, the cytochrome p450 3A familyis responsible for the metabolism of a majority of drugs. As shown inFIG. 22, primary rat hepatocytes cultured under controlled hemodynamicsin the devices with healthy physiological levels (n=6) of insulin (2nMol) and glucose (5.5 mMol) in the media, exhibit a significantlyhigher expression level of the key metabolic enzyme cytochrome p450 3a2(Cyp3A2; p=0.03), compared to those cultured under pathologicalconditions (n=9) with high insulin (2 μMol) and high glucose (17.5 mMol)levels. Both the healthy and pathological fatty liver levels undercontrolled flow are many fold higher than static cultures in collagengel sandwiches (2 μM insulin and 17.5 mMol glucose).

Similarly, as shown in FIG. 29A, expression of a number of phase Ienzymes involved in drug metabolism are differentially regulated underlow and high glucose/insulin conditions. Under hemodynamic flow,hepatocytes under healthy media conditions maintained high levels ofmRNA expression of Cyp1a1, Cyp 2b1, 2b2, Cyp3a2, and (20, 90, 30 and40-fold higher than traditional static cultures respectively), whereasCyp 2b2 and Cyp 3a2 levels in hepatocytes cultured in the fatty livermedia were decreased by 9 and 12 fold compared to healthy.

Cyp Activity: As shown in FIG. 29B, the activities of CYP3A2 and CYP1A1were also reduced 3-6-fold under the high insulin/glucose fatty liverconditions compared to healthy, as measured by the p45glo assay.

Pioglitazone treatment: Pioglitazone, a drug used to treat steatosis,was tested in the fatty liver model to determine if it could reverse thelipid accumulation and metabolic changes induced by the highinsulin/glucose fatty liver media. The pioglitazone was added to themedia at a concentration of 1.5 μM, a concentration selected based onthe therapeutic C_(max) observed for pioglitazone in vivo. Pioglitazonewas effective in reducing the lipid buildup and triglyceride contentwhile restoring metabolic gene expression under the disease conditions.As shown in FIGS. 30A-30C, Nile red staining indicates that treatmentwith pioglitazone at in vivo therapeutic concentrations decreases lipiddroplet formation under steatotic conditions. Pioglitazone also reducedtotal triglyceride content of hepatocytes cultured in the highinsulin/glucose media to levels similar to those seem in the hepatocytescultured under healthy conditions (FIG. 31). Moreover, as shown in FIG.32, pioglitazone restored the expression of metabolic genes such asCyp3A2 which are depressed by the high insulin/glucose diseaseconditions.

Conclusions:

In summary, a system was developed that preserves in vivo-likehepatocyte phenotype and response, to create a model of hepaticsteatosis by inducing pathological steatotic changes in the presence ofa high glucose/insulin milieu. Rat hepatocytes under controlledhemodynamics retain their response to insulin and glucose, andhepatocytes cultured under hemodynamic flow develop steatotic changeswhen cultured in high glucose and insulin (‘disease’) conditions. Thesteatosis is mediated via de novo lipogenesis with upregulation of twokey genes (SREBP and GPAT), and the increase in lipid accumulation andtriglyceride content is accompanied by a concomitant decrease inmetabolic gene expression and activity. Treatment with the PPAR-γagonist pioglitazone helps prevent the buildup of lipid and loss ofmetabolic activity under the high glucose and insulin conditions. Thesedata demonstrate a novel and important new in vitro model of dietinduced non-alcoholic fatty liver disease (NAFLD) for which nonecurrently exist.

When introducing elements of the present invention or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods without departingfrom the scope of the invention, it is intended that all mattercontained in the above description and shown in the accompanyingdrawing[s] shall be interpreted as illustrative and not in a limitingsense.

1-299. (canceled)
 300. A method of mimicking a pathological orphysiologic condition in vitro, the method comprising: adding a culturemedium and at least one factor to a cell culture container; plating atleast one cell type on at least one surface within the cell culturecontainer, wherein the surface comprises a surface of a porous membranesuspended in the cell culture container; and applying a shear force upona second surface of the porous membrane, the shear force resulting fromflow of the culture medium induced by a flow device, the flow mimickingflow to which the at least one cell type is exposed in vivo in thepathological or physiologic condition, wherein: the concentration of thefactor in the culture media for mimicking the pathological condition iseither: (i) within the in vivo concentration range of the factorobserved in the pathological condition; or (ii) within the concentrationrange of the factor that would result in vivo from administration of adrug or a compound; or the concentration of the factor in the culturemedia for mimicking the physiologic condition is either: (i) within thein vivo concentration range of the factor observed in the physiologiccondition; or (ii) within the concentration range of the factor thatwould result in vivo from administration of a drug or a compound. 301.The method of claim 300, wherein the method further comprises testing adrug or a compound for an effect on the pathological or physiologiccondition, wherein testing the drug or the compound for the effect onthe pathological or physiologic condition comprises: adding the drug orthe compound to the culture medium after plating the at least one celltype; and applying the shear force upon the at least one cell typeexposed to the drug or the compound; wherein a change in the at leastone cell type in the presence of the drug or the compound indicates thatthe drug or the compound has an effect on the pathological orphysiological condition.
 302. The method of claim 300, wherein thepathological condition is mimicked.
 303. The method of claim 302,wherein the concentration of the factor in the culture media is withinthe in vivo concentration range of the factor observed in thepathological condition.
 304. The method of claim 302, wherein theconcentration of the factor in the culture media is within theconcentration range of the factor that would result in vivo fromadministration of a drug or a compound.
 305. The method of claim 300,wherein a change in a level of a marker of the pathological condition inthe at least one plated cell type or in the culture media uponapplication of the shear force, as compared to the level of the markerin the at least one plated cell type or in the culture media in theabsence of application of the shear force confirms mimicking of thepathological condition.
 306. The method of claim 300, wherein thephysiologic condition is mimicked.
 307. The method of claim 306, whereinthe concentration of the factor in the culture media is within the invivo concentration range of the factor observed in the physiologiccondition.
 308. The method of claim 306, wherein the concentration ofthe factor in the culture media is within the concentration range of thefactor that would result in vivo from administration of a drug or acompound.
 309. The method of claim 300, wherein a change in a level of amarker of the physiologic condition in the at least one plated cell typeor in the culture media upon application of the shear force, as comparedto the level of the marker in the at least one plated cell type or inthe culture media in the absence of application of the shear forceconfirms mimicking of the physiologic condition.
 310. The method ofclaim 300, wherein the at least one plated cell type comprises renalcells, cells of the airways, blood-brain barrier cells, vascular cells,hepatic cells, pancreatic cells, cardiac cells, muscle cells, spleencells, gastrointestinal tract cells, skin cells, liver cells, immunecells, or hematopoietic cells.
 311. The method of claim 300, wherein theat least one plated cell type comprises astrocytes, endothelial cells,glomerular fenestrated endothelial cells, renal epithelial podocytes,alpha cells, β-cells, delta cells, pancreatic polypeptide (PP) cells,epsilon cells, glial cells, hepatocytes, neurons, nonparenchymal hepaticcells, podocytes, smooth muscle cells, mesangial cells, pericytes,cardiac muscle cells, skeletal muscle cells, leukocytes, monocytes,myocytes, macrophages, neutrophils, dendritic cells, T-cells, B-cells,endothelial progenitor cells, stem cells, circulating stem cells,circulating hematopoietic cells, or a combination of any thereof. 312.The method of claim 300, wherein the pathological condition comprises avascular pathological condition, the factor comprises oxidizedlow-density lipoprotein (oxLDL), tumor necrosis factor-α (TNFα),glucose, tissue growth factor-β (TGF-β), an elastin degradation product,elastase, vitamin D, an inorganic phosphate, leptin, adiponectin,apelin, aldosterone, angiotensin II, a triglyceride, high-densitylipoprotein (HDL), oxidized high-density lipoprotein (oxHDL), atriglyceride-rich lipoprotein, low-density lipoprotein (LDL), insulin, afatty acid, or a combination of any thereof.
 313. The method of claim312, wherein the at least one cell type comprises endothelial cells,smooth muscle cells, or endocardial cells.
 314. The method of claim 300,wherein the factor comprises TNFα, a fatty acid, or a combinationthereof.
 315. The method of claim 302, wherein the pathologicalcondition comprises advanced inflammation, atherosclerosis, diabeticnephropathy, diabetic neuropathy, diabetic retinopathy, hypertension,hypertensive encephalopathy, hypertensive retinopathy, fatty liverdisease, hypertension, heart failure, stroke, Marfan syndrome, carotidintima-medial thickening, atrial fibrillation, kidney disease, pulmonaryfibrosis, chronic obstructive pulmonary disease, hyperlipidemia,hypercholesterolemia, diabetes, atherosclerotic plaque rupture,atherosclerotic plaque erosion, thoracic aortic aneurysm, cerebralaneurysm, abdominal aortic aneurysm, cerebral aneurysm, pulmonary arterydisease, pulmonary hypertension, peripheral artery disease, deep veinthrombosis, vascular restenosis, vascular calcification, myocardialinfarction, obesity, hypertriglyceridemia, hypoalphalipoproteinemia,hepatitis C, hepatitis B, liver fibrosis, bacterial infection, viralinfection, cirrhosis, or alcohol-induced liver disease.
 316. The methodof claim 300, wherein plating the at least one cell type on the surfaceof the porous membrane comprises plating a first cell type on a firstsurface of the porous membrane and plating a second cell type on asecond surface of the porous membrane, wherein the shear force isapplied upon the second cell type.
 317. The method of claim 316,wherein: the first cell type comprises smooth muscle cells and thesecond cell type comprises endothelial cells; or the first cell typecomprises hepatocytes and the second cell type comprises non-parenchymalhepatic cells.
 318. The method of claim 300, wherein: the porousmembrane is positioned in the cell culture container such that a firstsurface of the porous membrane forms a boundary of a first volume withinthe container and the second surface forms a boundary of a second volumewithin the container; the first volume comprises the at least one celltype; and the shear force is applied by inducing the flow of the culturemedium within the second volume of the container.
 319. An in vitromethod of testing a drug or a compound for an effect, the methodcomprising: adding a culture media to a cell culture container; platingat least one cell type on at least one surface within the cell culturecontainer; adding a drug or a compound to the culture media, wherein theconcentration of the drug or the compound in the culture media is withinthe concentration range of the drug or the compound that achieves theeffect in vivo; and applying a shear force upon the at least one platedcell type exposed to the drug or the compound, the shear force resultingfrom flow of the culture media induced by a flow device, the flowmimicking flow to which the at least one cell type is exposed in vivo,wherein a change in the at least one plated cell type, in the presenceof the drug or the compound, indicates that the drug or the compound hasthe effect.